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ACTIVATION OF THE TRKB

NEUROTROPHIN RECEPTOR BY

ANTIDEPRESSANT DRUGS

HANNA ANTILA

Neuroscience Center

&

Division of Pharmacology and Pharmacotherapy

Faculty of Pharmacy

University of Helsinki

&

Doctoral Programme Brain & Mind

ACADEMIC DISSERTATION

To be presented for public examination with the permission of the Faculty of

Pharmacy of the University of Helsinki at University of Helsinki Main Building,

Auditorium XII, on 14th of September 2016 at 12 o’clock noon

Supervisors Docent Tomi Rantamäki, PhD

Department of Biosciences


Finland

Professor Eero Castrén, MD, PhD

Neuroscience Center


Finland

Reviewers Associate professor Annakaisa Haapasalo, PhD

Department of Neurobiology

University of Eastern Finland

Finland

Docent Mikko Airavaara, PhD (pharm.)

Institute of Biotechnology


Finland

Opponent Professor Moses Chao, PhD

Skirball Institute of Biomolecular Medicine

New York University Langone Medical Center

New York, United States of America

Custos Professor Raimo Tuominen, MD, PhD

Division of Pharmacology and Pharmacotherapy

Faculty of Pharmacy


Finland

ISBN 978-951-51-2424-1 (paperback)

ISBN 978-951-51-2425-8 (PDF)

ISNN 2342-3161 (paperback)

ISNN 2342-317X (PDF)

Hansaprint

Helsinki, Finland 2016

In memory of

Mumma and Matti

CONTENTS Abstract

Tiivistelmä

Abbreviations

List of original publications

1. INTRODUCTION ............................................................................................................................................. 1

2. REVIEW OF THE LITERATURE ................................................................................................................... 2

2.1 BDNF AND TRKB ..................................................................................................................................... 2

2.1.1 Discovery of neurotrophins ............................................................................................................... 2

2.1.2 Bdnf gene structure and regulation .................................................................................................. 2

2.1.3 Expression and localization of BDNF ............................................................................................... 4

2.1.4 Processing and secretion of BDNF protein ....................................................................................... 5

2.1.5 TrkB gene and mRNA ........................................................................................................................ 7

2.1.6 Functional domains and post-transcriptional processing of TrkB .................................................. 8

2.1.7 Expression and subcellular localization of TrkB .............................................................................. 9

2.1.8 TrkB activation by BDNF and downstream signaling ..................................................................... 11

2.1.9 Signaling of the truncated TrkB receptor ....................................................................................... 14

2.1.10 TrkB transactivation ...................................................................................................................... 14

2.1.11 ProBDNF and p75NTR signaling ...................................................................................................... 16

2.2 Role of BDNF and TrkB in CNS development and function ................................................................. 18

2.2.1 Cell survival and differentiation ...................................................................................................... 18

2.2.2 Plasticity........................................................................................................................................... 19

2.2.3 BDNF and TrkB mutations and polymorphisms in humans ......................................................... 22

2.3 NEUROTROPHIN AND NETWORK THEORIES OF ANTIDEPRESSANT ACTION ......................... 24

2.3.1 Antidepressant drugs ....................................................................................................................... 24

2.3.2 Concept of neurotrophin theory of depression and antidepressant action .................................. 24

2.3.3 Regulation of BDNF and TrkB by stress and antidepressant drugs .............................................. 25

2.3.4 Neurogenesis and depression ......................................................................................................... 26

2.3.5 The concept of network theory of depression and antidepressant action ..................................... 27

2.3.6 Plasticity models and antidepressant drug action ......................................................................... 28

2.4 RAPID-ACTING ANTIDEPRESSANT DRUGS ..................................................................................... 32

2.4.1 Short history of rapid antidepressant effects .................................................................................. 32

2.4.2 The effects of rapid-acting antidepressant ketamine ..................................................................... 32

2.4.3 The effects of other rapid-acting antidepressant drugs ................................................................. 36

3. AIMS OF THE STUDY ................................................................................................................................... 37

4. MATERIALS AND METHODS ..................................................................................................................... 38

4.1 Animals .................................................................................................................................................... 38

4.2 Drug treatments ...................................................................................................................................... 38

4.3 Cell culture .............................................................................................................................................. 38

4.3.1 Fibroblasts ........................................................................................................................................ 38

4.3.2 Primary neuronal cultures .............................................................................................................. 38

4.4 Enzyme-linked immunosorbent assay (ELISA) .................................................................................... 39

4.4.1 Conventional pTrkB ELISA ............................................................................................................. 39

4.4.2 In situ TrkB ELISA .......................................................................................................................... 39

4.5 Proof-of-concept small molecule screening ........................................................................................... 39

4.6 Brain sample collection ..........................................................................................................................40

4.7 Ex vivo stimulations ................................................................................................................................40

4.8 Western blot ............................................................................................................................................40

4.9 SDS-PAGE zymography .......................................................................................................................... 41

4.10 Immunohistochemistry and dendritic spine analysis ......................................................................... 41

4.11 Quantitative real-time polymerase chain reaction (qPCR) .................................................................. 41

4.12 Behavioral experiments ........................................................................................................................ 42

4.12.1 Forced swim test ............................................................................................................................ 42

4.12.2 Open field test ................................................................................................................................ 42

4.12.3 Water maze .................................................................................................................................... 42

4.13 Statistical tests ....................................................................................................................................... 42

5.RESULTS ........................................................................................................................................................ 43

5.1 Development of phospho-Trk ELISAs (I) ............................................................................................... 43

5.2 Mechanisms of antidepressant induced TrkB activation - BDNF and serotonin transporter (SERT) are

dispensable for TrkB activation by antidepressant drugs (II) .................................................................... 44

5.3 Developmental regulation of TrkB activation by antidepressant drugs and BDNF (III) ..................... 45

5.4 Isoflurane activates TrkB signaling, enhances synaptic plasticity and induces antidepressant-like

behavior (IV) ................................................................................................................................................. 46

6. DISCUSSION ................................................................................................................................................. 49

7. CONCLUSIONS ............................................................................................................................................. 55

ACKNOWLEDGEMENTS ................................................................................................................................. 56

REFERENCES ................................................................................................................................................... 58

ABSTRACT

Major depressive disorder is one of the most significant causes of disability worldwide.

Currently, the main treatment options for depression are psychotherapy and

antidepressant drugs that pharmacologically target the monoamine systems – such as

serotonin transporter (SERT) blocker fluoxetine. It has been hypothesized that

impairments in synaptic function and plasticity, caused for example by stress, could

underlie the manifestation of depression. Moreover, in rodent models chronic treatment

with antidepressant drugs has been shown to enhance plasticity of the adult brain via

brain-derived neurotrophic factor (BDNF). The effects of BDNF mediated via its receptor

tropomyosin receptor kinase B (TrkB) promote synapse function and thus could facilitate

recovery from depression. Interestingly, antidepressant drugs with different main

pharmacological targets seem to share the ability to activate the TrkB receptor, however,

the mechanisms how antidepressant drugs activate TrkB are not known.

The delayed onset of action and limited therapeutic efficacy of antidepressant drugs has

promoted interest toward finding more rapid-acting and effective treatment options for

depression. Electroconvulsive therapy has been the treatment of choice for treatment

resistant depressed patients, however, side effects and the disrepute among general

public has limited its use. Recently, subanesthetic doses of dissociative anesthetic

ketamine have been shown to rapidly alleviate depression symptoms in depressed

patients who do not respond to conventional antidepressant drugs. The effects of

ketamine on mood appear already couple of hours after single intravenous infusion and

last for about one week. Ketamine has been shown to induce mammalian target of

rapamycin (mTOR) via BDNF-TrkB signaling, rapidly promote synaptogenesis and alter

neural network function. Furthermore, in small human studies another anesthetic

isoflurane has rapidly alleviated symptoms of depressed patients. Yet, the potential of

isoflurane in the treatment of depression has not been studied in large clinical trials.

Since TrkB receptor is involved in regulation of synaptic plasticity, drugs that act as

agonists or positive allosteric modulators of TrkB could be potentially beneficial in the

treatment of CNS disorders characterized by impaired plasticity. The first aim of our

studies was to develop a platform suitable for high-throughput screening of compounds

regulating TrkB activity. We developed an in situ ELISA (enzyme-linked immunosorbent

assay) method that detects phosphorylated TrkB receptors from cultured cells. The main

advantage of the in situ ELISA compared to conventional ELISA is that the cells are

cultivated directly on the ELISA plate making the additional transfer step of the cellular

material from the cell culture plate to the ELISA plate unnecessary. To further validate

the in situ ELISA method, we conducted a proof-of-concept screening of a small chemical

library and found several compounds that dose-dependently activated TrkB receptor or

inhibited BDNF-induced TrkB activation.

The second aim was to examine the mechanism how the antidepressant drugs activate

TrkB. Interestingly, we found that antidepressant drugs activate TrkB independently of

BDNF. Moreover, SERT, the main pharmacological target of fluoxetine, was not required

for the fluoxetine-induced TrkB activation. Furthermore, the antidepressant-induced

TrkB activation was developmentally regulated. The ability of antidepressants to activate

TrkB appeared around postnatal day 12. Interestingly, at this same developmental

timepoint (P12) the ability of BDNF to activate TrkB decreased dramatically.

Finally, we aimed to characterize the neurobiological basis for the possible

antidepressant effects of isoflurane. We found that brief isoflurane anesthesia rapidly

and transiently activated the TrkB-mTOR signaling and produced antidepressant-like

behavioral response in the forced swim test in a TrkB-dependent manner. Single

isoflurane treatment also produced an antidepressant-like phenotype in behavioral

paradigms that normally require chronic treatment with conventional antidepressant

drugs, suggesting that isoflurane may have rapid antidepressant effects similar to

ketamine. Moreover, isoflurane facilitated hippocampal long-term potentiation when

measured 24 hours after the treatment and affected the general neural network function

by increasing activity of the parvalbumin-positive inhibitory interneurons in the

hippocampus.

In conclusion, our results improve the understanding of the mechanism of action of

conventional antidepressant drugs and provide plausible neurobiological basis for the

antidepressant effects of isoflurane. Our findings also support examining further the

potential of anesthetics in the treatment of depressed patients who do not respond to the

current treatment options.

TIIVISTELMÄ

Masennus on merkittävä kansanterveydellinen ongelma, jonka kehittymiseen on

ehdotettu liittyvän esimerkiksi pitkäkestoisen stressin aiheuttamia häiriöitä aivojen

muovautuvuudessa ja hermosoluyhteyksien toiminnassa. Masennuksen hoito perustuu

pääasiassa psykoterapiaan ja masennuslääkkeisiin. Kaikki kliinisessä käytössä olevat

masennuslääkkeet vaikuttavat aivojen monoaminergisiin järjestelmiin, ja

masennuslääkkeiden vaikutusten onkin pitkään ajateltu välittyvän yksinomaan näiden

järjestelmien kautta. Eläinkokeissa masennuslääkkeiden on havaittu voimistavan

aivojen muovautuvuutta aivoperäisen hermokasvutekijän (BDNF) välityksellä. BDNF

osallistuu hermoyhteyksien toiminnan säätelyyn TrkB (tropomyosin receptor kinase B)

–reseptorin välityksellä ja masennuslääkkeiden vaikutukset BDNF-TrkB –signalointiin

saattavatkin osaltaan edesauttaa masennuksesta toipumista. Kyky aktivoida TrkB-

reseptoria vaikuttaisikin olevan yhteinen ominaisuus muutoin eri kohdemolekyyleihin

vaikuttavilla masennuslääkkeillä. Tarkempaa mekanismia masennuslääkkeiden

aikaansaaman TrkB-reseptorin aktivaation taustalla ei kuitenkaan vielä tunneta.

Osa masennuspotilaista ei riittävästi hyödy nykyisistä masennuslääkkeistä ja

masennuslääkkeiden terapeuttiset vaikutukset ilmenevät viiveellä. Tämän vuoksi

masennuksen hoitoon yritetään jatkuvasti kehittää uusia, tehokkaampia ja nopeammin

toimivia lääkkeitä. Sähköhoito (ECT) on tällä hetkellä käytössä olevista hoitomuodosta

tehokkain, mutta se aiheuttaa muistihäiriöitä ja sen käyttöä rajoittavat lisäksi

voimakkaat ennakkoluulot. Viime aikoina nukutusaine ketamiinin on havaittu nopeasti

(muutamassa tunnissa) lievittävän masennusoireita muihin hoitoihin reagoimattomilla

potilailla. Ketamiinin masennusta lievittävien vaikutusten taustalla on esitetty olevan

sen kyky aktivoida BDNF-TrkB-mTOR (mammalian target of rapamycin) –signalointia,

nopeasti lisätä uusien hermosoluyhteyksien määrää ja muuttaa hermoverkkojen

toimintaa. Ihmisillä tehdyissä tutkimuksissa on lisäksi havaittu toisen nukutusaineen,

isofluraanin, lievittävän masennusoireita nopeasti. Isofluraanin tehoa masennuksen

hoidossa ei ole kuitenkaan vielä tutkittu laajemmissa kliinisissä tutkimuksissa.

TrkB-reseptorin välittämiä plastisuusvaikutuksia voitaisiin mahdollisesti hyödyntää

myös muiden keskushermostosairauksien kuin masennuksen hoidossa.

Tarkoituksenamme olikin kehittää menetelmä, jonka avulla voitaisiin seuloa TrkB-

reseptoriin vaikuttavia uusia molekyylejä. Kehitimme in situ ELISA (enzyme-linked

immunosorbent assay) –menetelmän, joka tunnistaa TrkB-reseptorin fosforyloituneen

eli aktivoituneen muodon solunäytteistä. In situ ELISA eroaa tavallisesta ELISAsta siten,

että solut kasvatetaan suoraan ELISA-levyllä. In situ ELISA soveltuu myös suurten

kirjastojen seulomiseen, koska siinä vältytään työläältä näytteiden siirrolta

soluviljelylevyltä ELISA-levylle. Osoittaaksemme menetelmän soveltuvuuden

seulomistarkoitukseen, seuloimme 2000 yhdisteen kirjaston ja identifioimme useita

Trk-reseptoria aktivoivia sekä BDNF:n vaikutuksia estäviä yhdisteitä.

Tämän jälkeen tutkimme, miten masennuslääkkeet saavat aikaan TrkB-reseptorin

aktivoitumisen hiiressä. Yllättäen havaitsimme, että masennuslääkkeet aktivoivat TrkB-

reseptorin ilman BDNF:ä, transaktivaation välityksellä. Lisäksi fluoksetiinin

aikaansaama TrkB-reseptorin aktivoituminen tapahtui ilman, että sen täytyi sitoutua

pääasialliseen kohdemolekyylinsä serotoniinitransportteriin (SERT).

Masennuslääkkeiden aikaansaama TrkB-reseptorin aktivoituminen oli myös

kehityksellisesti säädeltyä ilmeten vasta 12 päivän ikäisillä hiirillä. Tässä samassa

kehitysvaiheessa BDNF:n aikaansaama TrkB-reseptorin aktivoituminen taas väheni

merkittävästi.

Lopuksi selvitimme, minkälaisten neurobiologisten prosessien kautta nukutusaine

isofluraanin mahdolliset masennusta lievittävät vaikutukset voisivat välittyä.

Tutkimuksemme osoittivat isofluraanin aktivoivan hiiressä TrkB-mTOR –signalointia ja

aiheuttavan pakotetussa uintitestissä (forced swim test) masennuslääkkeen kaltaisen

käyttäytymisvasteen, joka välittyi TrkB-reseptorin kautta. Lisäksi yksi isofluraani-

nukutus sai aikaan masennuslääkkeen kaltaisen käyttäytymisvasteen testeissä, jotka

normaalisti vaativat pitkäaikaisen käsittelyn masennuslääkkeillä. Tämä osoittaakin, että

isofluraani saattaisi toimia ketamiinin tapaan nopeavaikutteisena masennuslääkkeenä.

Kestotehostuminen (LTP, long-term potentiation) voimistui ja parvalbumiinia

ilmentävien estävien välineuronien aktiivisuus lisääntyi hippokampuksessa 24 tuntia

isofluraani-käsittelyn jälkeen, osoittaen että yhdellä nukutuksella on pitkäkestoisia

vaikutuksia myös hermoverkkojen toimintaan.

Tutkimustuloksemme tuovat lisää tietoa masennuslääkkeiden vaikutusmekanismeista ja

voivat selittää, minkä vuoksi isofluraanilla saattaa olla masennusoireita lievittäviä

vaikutuksia. Lisäksi tulostemme perusteella nukutusaineiden käyttökelpoisuutta muihin

hoitoihin reagoimattomien masennuspotilaiden hoidossa kannattaisi tutkia lisää.

ABBREVIATIONS

5-HT 5-hydroxytryptamine, serotonin AD Antidepressant drug AKT Protein kinase B AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid BDNF Brain-derived neurotrophic factor BrdU Bromodeoxyuridine CA Cornu ammonis (in hippocampus) CaMKII Calcium/calmodulin-dependent protein kinase II CNS Central nervous system CREB Cyclic AMP response element-binding protein DAG Diacylglycerol DNA Deoxyribonucleic acid ECT Electroconvulsive therapy eEF2 Eucaryotic elongation factor 2 EGF Epidermal growth factor ELISA Enzyme-linked immunosorbent assay ERK Extracellular signal-regulated kinase FST Forced swim test GABA Gamma-aminobutyric acid GPCR G-protein coupled receptor GSK3β Glycogen synthase kinase 3 beta HFS High frequency stimulation HNK Hydroxynorketamine IgG Immunoglobulin G IP3 Inositol trisphosphate LRR Leucine-rich repeat LTD Long-term depression LTP Long-term potentiation MAO Monoamine oxidase MMP Matrix metalloproteinase mRNA Messenger ribonucleic acid mTOR Mammalian target of rapamycin NBQX 2,3- Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-

sulfonamide NGF Nerve growth factor NMDA N-methyl-D-aspartate NT-3 Neurotrophin 3 NT-4 Neurotrophin 4 PACAP Pituitary adenylate cyclase-activating polypeptide p75NTR P75 neurotrophin receptor PC Pro-convertase PFC Prefrontal cortex PI3k Phosphoinositide 3-kinase PKC Protein kinase C PLCγ Phospholipase C-gamma PNS Peripheral nervous system PP1 Protein phosphatase 1 proBDNF Pro-form of Brain-derived neurotrophic factor qPCR Quantitavive real-time polymerase chain reaction Shc Src-homology 2 domain-containing SERT Serotonin transporter SGZ Subgranular zone of hippocampus SSRI Selective serotonin reuptake inhibitor SVZ Subventricular zone tPA Tissue plasminogen activator Trk Tropomyocin receptor kinase VEP Visually evoked potential VTA Ventral tegmental area Y Tyrosine

LIST OF ORIGINAL PUBLICATIONS

This thesis is based on the following publications:

I Antila H, Autio H, Turunen L, Harju K, Tammela P, Wennerberg K, Yli-

Kauhaluoma J, Huttunen H, Castrén E, Rantamäki T: Utilization of in situ

ELISA method for examining TrkB receptor phosphorylation in cultured cells. J

Neurosci Methods, 2013 Nov 12;222C,142-146

II Rantamäki T, Vesa L*, Antila H*, di Lieto A, Tammela P, Schmitt A, Lesch

KP, Rios M, Castrén E: Antidepressant drugs transactivate TrkB neurotrophin

receptors in the adult rodent brain independently of BDNF and monoamine

transporter blockade. PloSOne 2011;6(6):e20567 *Equal contribution

III Di Lieto A, Rantamäki T, Vesa L, Yanpallewar S, Antila H, Lindholm J,

Rios M, Tessarollo L, Castrén E: The responsiveness of TrkB to BDNF and

antidepressant drugs is differentially regulated during mouse development.

PLoS One 2012;7(3):e32869

IV Antila H, Casarotto P, Popova D, Sipilä P Guirado R, Kohtala S,

Ryazantseva M, Vesa L, Lindholm J, Yalcin I, Sato V, Nurkkala H, Lemprière S,

Cordeira J, Autio H, Kislin M, Rios M, Joca S, Khiroug L, Lauri S, Varjosalo M,

Grant SGN, Taira T, Castrén E, Rantamäki T : TrkB signaling underlies the

rapid antidepressant effects of isoflurane. Submitted manuscript.

1

1. INTRODUCTION

Major depression is the largest contributor to the worldwide disease burden when

measured as years lost to disability. This occurs primarily because depression can persist

for many years and a large number of individuals (~350 million) suffer from it (Smith,

2014). Antidepressant drugs (AD) and psychotherapy are the main treatment options for

depression, however, significant amount of depressed patients do not respond to the

treatment.

It has been suggested that stress-induced changes in neuronal connectivity and resulting

disturbances in network function are important factors in the pathophysiology of

depression (Castrén & Hen, 2013). Brain-derived neurotrophic factor (BDNF) and its

receptor tropomyosin receptor kinase B (TrkB) are known to regulate neuronal plasticity,

pathology of depression and the mechanism of action of ADs. ADs promote the

expression of BDNF and activate its receptor TrkB (Nibuya et al., 1995a; Rantamäki et

al., 2007; Saarelainen et al., 2003). Since BDNF and TrkB are involved in the regulation

of neuronal excitability, cell survival and plasticity, the ability of ADs to increase their

expression has been suggested to underlie the therapeutic effects of ADs.

However, delayed onset of action and poor efficacy of ADs limits their therapeutic use.

Significant attempts to find novel drugs for treatment of depression have been

conducted. Electroconvulsive therapy (ECT) remains the treatment of choice for patients

unresponsive to multiple trials with different ADs and psychotherapy. Recently,

however, a subanesthetic dose of ketamine has been shown to rapidly alleviate

depression symptoms and to reduce suicidal ideation in treatment resistant depressed

(TRD) patients and BDNF-TrkB signaling has been shown to be involved in these

therapeutic effects of ketamine (Autry et al., 2011; Berman et al., 2000; Zarate CA et al.,

2006). Ketamine, however, may produce hallucinogenic effects and has significant abuse

potential, thus it is not an optimal drug to replace the conventional antidepressant

treatments. Intriguingly, volatile anesthetic isoflurane has been shown to relieve

depression symptoms of TRD patients as effectively as ECT but without the cognitive

side effects characterized with ECT (Langer et al., 1985, 1995; Weeks et al., 2013).

Altogether these preliminary findings encourage investigating further the antidepressant

potential of anesthetics in animal models and human patients.

In this thesis the role of BDNF and TrkB in the central nervous system and in the effects

of antidepressant drugs will be discussed. In the experimental section we have developed

tools to screen for novel TrkB activators, investigated the mechanisms of antidepressant-

induced TrkB activation, and dissected the neurobiological basis for the antidepressant

effects of isoflurane.

2

2. REVIEW OF THE LITERATURE

2.1 BDNF AND TRKB

2.1.1 Discovery of neurotrophins

The discovery of the first neurotrophin, the nerve growth factor (NGF), and the

characterization of its effects on the survival and target innervation of subpopulation of

neurons in the peripheral nervous system (PNS) were done by Rita Levi-Montalcini,

Victor Hamburger and Stanley Cohen (Levi-Montalcini, 1987). These findings were

seminal to the idea that the non-neural target tissue secretes factors that affect the

survival of neurons innervating it; a concept nowadays known as the neurotrophic

hypothesis (Bothwell, 2014). According to the hypothesis, now supported by massive

amounts of experimental data, neurotrophins are released from the target tissue in very

limited amounts allowing the survival of only a small number of neurons during early

development (Bothwell, 2014).

NGF alone was not sufficient to understand all of the neurotrophic effects detectable

during early development, thus brain-derived neurotrophic factor (BDNF) was

discovered. As the name implies, BDNF was first extracted from pig brain tissue (Barde

et al., 1982), indicating that neurotrophins also act at the level of central nervous system

(CNS). The characterization of the other members of the neurotrophin family -

Neurotrophin-3 (NT-3) and Neurotrophin 4 (NT-4) - was facilitated by the technical

development in molecular biology, especially the discovery of polymerase chain reaction

(PCR), since the gene structures of already known neurotrophins could be exploited to

find similar proteins (Hallböök et al., 1991; Maisonpierre et al., 1990a). Currently the

mammalian neurotrophin family consists of four structurally and functionally similar

members: NGF, BDNF, NT-3 and NT-4.

The signaling effects of neurotrophins are mediated via the p75 neurotrophin receptor

(p75NTR) and the Trk receptor tyrosine kinases. All the neurotrophins can activate the

signaling via p75NTR, but their binding affinities to the Trk receptors are more specific:

NGF binds to TrkA, BDNF and NT-4 to TrkB and NT-3 to TrkC (Klein et al., 1991a, 1991b,

1992; Lamballe et al., 1991).

Although all neurotrophins act as target-derived survival factors in the PNS during the

development, their functions in the CNS appear much more diverse and complex. The

literature review focuses on BDNF since it is the most abundant neurotrophin in the

brain and it has been linked with the pathophysiology of depression, mechanism of

action of antidepressant drugs and brain plasticity. These issues and the basic

neurobiology of BDNF and TrkB will be introduced in the subsequent sections.

2.1.2 Bdnf gene structure and regulation

The bdnf gene consists of eight 5’ non-coding, regulatory exons and one 3’ coding exon

(exon IX) (Aid et al., 2007) (Fig 1A). The complexity of the bdnf gene allows precise

temporal and spatial regulation of BDNF expression. The exons are controlled by distinct

promoters that can be differentially regulated. Importantly however, all the transcripts

eventually encode the same BDNF protein. Various stimuli can activate different

transcription factors, which can then bind to different promoter regions of the bdnf exons

3

resulting in transcription of specific bdnf transcripts (West et al., 2014). Furthermore,

the transcripts have distinct localization, stability and translational regulation inside the

cell. Deletion of promoter regions IV and VI from the bdnf gene results in a robust

reduction of bdnf expression in the hippocampus and prefrontal cortex (PFC), whereas

deletion of promoter regions I and II reduces BDNF expression in the hypothalamus

(Maynard et al., 2015). These studies support the area specific roles of the bdnf

promoters.

Fig 1. A Structure of the bdnf gene (modified from Aid et al. 2007). The grey are represents the protein coding region. B Structure of the BDNF protein showing the different domains and the N-

glycosylation site.

The distinct effects of the individual bdnf transcripts is further supported by the altered

behavior and serotonergic functions of mice in which BDNF production is disrupted from

the promoters I, II, IV or VI (Maynard et al., 2015). For example, exon I and II specific

knock out mice show increased aggressive behavior whereas mice lacking exon IV or VI

do not. It has been previously shown however, that bdnf deletion from the ventromedial

and dorsomedial hypothalamus does not cause aggressive behavior (Unger et al. 2007),

suggesting that the effects of BDNF on hypothalamic circuits regulating aggression are

derived from developmental abnormalities in the network formation. Exon I knockouts

show increased expression of the serotonin (5-HT) transporter (SERT), the 5HT2A

receptor and parvalbumin in the prefrontal cortex (Maynard et al., 2015). Exon IV and

VI knockouts have reduced gene expression of markers for GABAergic interneurons, e.g.

parvalbumin (only in exon IV knockout mice), cortistatin and somatostatin in the PFC.

Neuronal activity strongly regulates bdnf transcription. Specifically the expressions of

bdnf exons I, II and IV are regulated in an activity-dependent manner (West et al., 2014).

Bdnf exon IV expression is strongly induced by elevations in intracellular calcium

concentration (Hong et al., 2008; Tao et al., 1998). Transcription factor cAMP response

element binding protein (CREB) is an important mediator of the activity- and calcium-

dependent transcription of bdnf (Chen and Russo-Neustadt, 2009; Tao et al., 1998).

Indeed, mutation in the CREB-binding site in the bdnf promoter IV impairs the activity-

dependent bdnf transcription (Hong et al., 2008). In addition, calcium-responsive

4

transcription factor (CaRF) regulates bdnf exon IV transcription in neurons in an

activity-dependent manner (Tao et al., 2002).

Potassium chloride and kainic acid are widely used agents to increase excitatory

neurotransmission and neuronal activity. Stimulation with potassium chloride or kainic

acid increased bdnf expression AMPAR- (α-amino-3-hydroxy-5-methyl-4-

isoxazolepropionic acid receptor) and calcium-dependently in cultured neurons (Zafra

et al., 1990). Even though NMDA (N-methyl-D-aspartate) receptors were not required

for these effects, NMDA receptor -mediated activity is important in more physiological

conditions since NMDA antagonists can prevent increases in bdnf messenger ribonucleic

acid (mRNA) normally observed during maturation in neuronal cultures (Zafra et al.

1991). Most importantly, sensory stimuli, or lack thereof, strongly regulate bdnf

transcription in vivo. For example, dark rearing decreases bdnf expression in the rat

visual cortex but re-exposure to light quickly restores bdnf mRNA levels (Castrén et al.,

1992). Moreover, increased neuronal activity following physical exercise, drug treatment,

or seizures increase bdnf transcription (Chen and Russo-Neustadt, 2009; Nibuya et al.,

1995a; Russo-Neustadt et al., 1999; Zafra et al., 1991). In contrast, pharmacologically-

induced neuronal inhibition or reduction of neuronal excitability decreases bdnf mRNA

levels in vivo (Zafra et al. 1991). Bdnf transcription also appears to be stress-responsive

since acute and chronic stress reduce hippocampal bdnf mRNA levels, while increasing

bdnf expression in the hypothalamus and pituitary (Smith et al., 1995a, 1995b).

2.1.3 Expression and localization of BDNF

BDNF protein is widely expressed in the brain, with highest levels detected in the

cerebral cortex and the hippocampus (Conner et al., 1997; Ernfors et al., 1992; Hofer et

al., 1990). The expression of BDNF was initially thought to be limited to neurons (Zafra

et al., 1990) but it is now widely accepted that brain microglia – particularly activated

microglia – can take up and release BDNF as well (Parkhurst et al., 2013). BDNF seems

to be expressed mainly in principal glutamatergic neurons but not in inhibitory

interneurons (Gorba and Wahle, 1999; Kuczewski et al., 2009). Although certain areas

of the brain essentially lack bdnf mRNA expression, transported BDNF protein can be

detected in these areas (Altar et al., 1997).

BDNF expression in the brain is strongly regulated during development. The expression

of bdnf mRNA gradually increases during early postnatal life, plateauing in rodents

around 3 weeks of age (Maisonpierre et al., 1990b; Rauskolb et al., 2010). In the human

dorsolateral prefrontal cortex (dlPFC) bdnf expression increases about one-third from

postnatal levels to adulthood peaking during early adulthood (around 22 years of age)

(Webster et al., 2002). Importantly, the peak of bdnf expression in the dlPFC is seen at

the age when the structural and functional maturation of the PFC occurs. In the

hippocampus bdnf mRNA levels seem to stay relatively constant during human life span,

including the aging brain (Webster et al., 2006).

In cultured neurons BDNF protein is found in the soma, as well as also in axons and in

dendrites (Adachi et al., 2005; Conner et al., 1997; Kohara et al., 2001). In vivo the

dendritic expression of bdnf mRNA has been demonstrated in apical dendrites of

hippocampal CA1 neurons (An et al., 2008; Tongiorgi et al., 2004). BDNF protein has

been primarily located in dense-core vesicles of the presynaptic terminals of excitatory

neurons (Dieni et al., 2012). These partially controversial findings of in vitro versus in

5

vivo BDNF localization may be explained by the differential and complex regulatory

processes of synapse formation and intracellular trafficking of neurons in the adult brain

compared to neuronal cultures.

The retrograde transport of neurotrophins in the PNS is the basis for the neurotrophic

hypothesis, where survival of the innervating neurons is regulated by the neurotrophin

released from the target tissue in a constitutive manner. Although the situation is more

complex in the brain, retrograde transport of BDNF has been demonstrated for example

in the eye, from where BDNF could be transported into the isthmo-optic nucleus (von

Bartheld et al., 1996). Striatal infusion of BDNF resulted in retrograde BDNF transport

to brain regions known to project to striatum e.g. thalamic areas and substantia nigra

pars compacta (Mufson et al., 1994). Application of BDNF to dendrites but not to axons

induced immediate early gene expression (c-fos, Arc) in the soma; indicating that BDNF-

induced signals from dendrites are conveyed to the soma (Cohen et al., 2011).

One of the first studies showing anterograde transport of BDNF was done by Zhou et al.

in primary sensory neurons (Zhou and Rush, 1996). BDNF was shown to be transported

both retrogradely and anterogradely, functioning as a modulator of synaptic

transmission or as a trophic factor for the organs that the neurons were innervating.

Kohara et al. (2001) demonstrated the anterograde axonal transport of BDNF in cultured

cortical neurons. Follow-up studies from the same group showed that the transport of

BDNF in axons is mainly anterograde, whereas in dendrites BDNF seemed to be not

moving at all or moving back and forth in a slower fashion (no clear retrograde transport)

(Adachi et al., 2005). In the striatum BDNF protein, but not mRNA is present; indicating

that BDNF is transported to the striatum anterogradely from the cortex by projection

neurons (Kolbeck et al., 1999). In the nucleus accumbens bdnf mRNA levels are low and

BDNF protein is transported there mainly from the ventral tegmental area (Altar et al.,

1997; Conner et al., 1997; Horger et al., 1999). In BDNF knockout animals the number of

parvalbumin-expressing neurons in the striatum was reduced at two weeks of age

suggesting that the BDNF transported to the area is regulating the maturation or survival

of this neuronal population (Altar et al., 1997). Noradrenergic neurons projecting to the

cortex were shown to express BDNF and transport it anterogradely to the cortex to

regulate the survival of the target neurons (Fawcett et al., 1998).

2.1.4 Processing and secretion of BDNF protein

BDNF is synthesized as pre-proBDNF, a precursor protein that is further processed in

the endoplasmic reticulum to proBDNF (Greenberg et al., 2009) (Fig 1B). The proBDNF

isoform is then N-glycosylated and glycosulfated (Mowla et al., 2001). The glycosylation

increases the stability of proBDNF during processing and subcellular trafficking. The

pro-domain participates in the proper folding and intracellular sorting of BDNF

(Brigadski et al., 2005; Lee et al., 2001b). The translated protein is further processed in

the Golgi and trans-Golgi network and directed to synaptic vesicles for release. The

proBDNF isoform can be cleaved to produce the mature form of BDNF (mBDNF) inside

the cell in trans-Golgi network or post-Golgi compartments by pro-convertases and

furin, or outside the cell by matrix metalloproteinases (MMP-7) or plasmin (Lee et al.,

2001b; Pang et al., 2004; Seidah et al., 1996). The efficacy of proBDNF cleavage to

mBDNF seems to vary during development. Postnatally and during adolescence both

proBDNF and mBDNF are expressed at similar levels, but in the adult brain the mature

form dominates. Furin is the main cleavage enzyme of the constitutive pathway guiding

6

the neurotrophins to the constitutive pathway (for example in fibroblasts) (Mowla et al.,

1999). The processing enzymes in the regulated and constitutive secretion pathways are

different and the cleavage of BDNF occurs in different subcellular compartments. In CNS

neurons BDNF is primarily directed to the regulated secretion pathway where furin is

not the essential enzyme for the processing of BDNF in the CNS neurons (Mowla et al.,

1999). In neurons the pro-convertases (PC) cleave the neurotrophin precursors inside

immature secretory vesicles in the trans-Golgi and are involved in the processing of

neurotrophins in the regulatory pathway. In the hippocampus and amygdala, PC7, an

enzyme of the proprotein convertase family, is involved in the intracellular processing of

proBDNF to BDNF (Wetsel et al., 2013).

In non-neuronal tissue BDNF appears to be constitutively released but in the brain the

secretion is mainly regulated through activity-dependent mechanisms (Mowla et al.,

1999). BDNF is stored in dense-core vesicles in the membrane fraction of synaptic

terminals (Fawcett et al., 1997). In vitro BDNF can be released both pre- and

postsynaptically and in cultured hippocampal neurons BDNF has been detected in

dendrites and axons (Adachi et al., 2005; Jakawich et al., 2010; Matsuda et al., 2009).

Interestingly pre- and postsynaptic release of BDNF seems to require different patterns

of stimulation. For example, the dendritic release of BDNF is dependent on calcium

influxes via NMDA receptors and L-type voltage-gated calcium channels. Furthermore,

BDNF can induce its own release via a positive feedback loop including stimulation of

TrkB receptors, activation of phospholipase C-gamma (PLCγ) and mobilization of

intracellular calcium stores (Canossa et al., 1997, 2001). Stimulation of metabotropic

glutamate receptors can also activate PLCγ and induce calcium release from intracellular

storages via inositol trisphosphate (IP3) resulting in BDNF release (Canossa et al., 2001).

Interestingly, the effect of glutamate on BDNF release can be blocked with AMPA

receptor antagonists but not NMDA receptor antagonists, and AMPA can increase BDNF

release from hippocampal slices and cultured neurons.

In vivo BDNF is co-expressed with the cleaved pro-peptide in dense core vesicles

presynaptically in the hippocampus, indicating that BDNF is cleaved inside the vesicles

and possibly released together with the cleaved pro-domain (Dieni et al., 2012). Since

there are enzymes that are able to cleave proBDNF to mBDNF also extracellularly, it has

been debated whether proBDNF can be released from the neurons or if it is processed to

mature BDNF in secretory vesicles before release. Lee et al. (2001b) suggested that

proBDNF is released from endothelial cells and can then be processed by tissue

plasminogen activator (tPA) and MMPs, and Chen et al. (2004) showed that proBDNF

is primarily released from the PC12 cells but is then quickly cleaved outside the cell. In

another study where proBDNF and mBDNF levels were measured in hippocampal

neuron cultures from the cell lysate and medium (secreted), the authors found that in

the presence of plasmin inhibitor α2-antiplasmin and in the culture conditions where

glial cell amount is reduced, proBDNF but not mBDNF is found in the medium,

supporting the importance of extracellular cleavage of secreted proBDNF (Yang et al.,

2009). When hippocampal neurons were infected with proBDNF expressing virus,

proBDNF was secreted into the medium and the amount of proBDNF in the medium

increased over time (Mowla et al., 1999). Also cultured cortical neurons were shown to

release proBDNF into the medium (Teng et al., 2005). In a study by Matsumoto et al.

(2008) the authors suggest that in hippocampal neurons endogenous proBDNF is

processed into BDNF already intracellularly (Matsumoto et al., 2008). Altogether the

7

release of BDNF/proBDNF has been mainly studied in overexpression systems or non-

neuronal cell lines using transfection of BDNF complementary DNA (cDNA), and

Matsumoto et al. suggest that in these situations the cells may lack the machinery for

proper processing of proBDNF or the capacity to process proBDNF to mature BDNF is

saturated, which could lead to biased results. Thus, it is difficult to reliably measure

BDNF secretion in vivo.

2.1.5 TrkB gene and mRNA

The BDNF receptor TrkB was first cloned from mouse brain tissue by virtue of its high

sequence homology to the NGF receptor TrkA (Klein et al., 1989). The TrkB gene

(NTRK2) is capable of producing multiple transcripts including the full-length catalytic

tyrosine kinase receptor but also TrkB receptor variants that lack the catalytic kinase

domain (Klein et al., 1990; Middlemas et al., 1991; Stoilov et al., 2002). The full-length

TrkB receptor consists of an extracellular ligand-binding domain, a transmembrane

anchoring domain and an intracellular domain that includes the highly conserved

catalytic kinase domain (Klein et al., 1990). The truncated TrkB receptors (TrkB.T1 and

TrkB.T2) share similar extracellular and transmembrane domains to the full-length

receptor but have only a short intracellular part consisting of a unique sequence of amino

acid residues. In the human brain there is no expression of TrkB.T2, however, an

additional isoform, TrkB.Shc, is present and lacks the tyrosine kinase domain but

contains the intracellular Shc site (Stoilov et al., 2002). In mouse a TrkB receptor isoform

lacking the extracellular leucine rich repeats completely or in part has been identified

(Ninkina et al., 1997).

The human TrkB gene is large, consisting of 24 exons that produce multiple mRNAs

which can produce up to 10 different protein isoforms (Stoilov et al., 2002) (Fig 2A).

Three isoforms, TrkB.FL, TrkB.T1 and TrkB.Shc are predominantly expressed at the

protein level. The exons 6-15 of the TrkB gene encode the extracellular domain, the

transmembrane domain, and a part of the juxtramembrane domain of the receptor. Exon

16 includes a stop codon and is part of the TrkB.T1. Exons 17-18 encode the intracellular

juxtramembrane part and exon 19 is an alternative terminating exon involved in the

TrkB.Shc. Exons 20-24 encode the tyrosine kinase domain and the PLCγ site of the

receptor.

8

Fig 2. A Exon structures of the three predominant TrkB isoforms: Full-length TrkB, TrkB.Shc lacking the tyrosine kinase domain but containing the Shc binding site and TrkB.T1 lacking the intracellular domains. B TrkB protein domains; C, cysteine rich region; Leu, leucine rich region; IG-L, immunoglobulin like-domain; TMD, transmembrane domain; Shc, Shc binding site; TKD, tyrosine kinase domain; PLCγ, PLCγ binding site. Modified from. Luberg et al. 2010.

In humans, additional N-terminal truncated TrkB receptors have been identified (Luberg

et al., 2010). These receptors lack the signal peptide that targets the receptors to the

membrane and also the leucine-rich repeats (LRR) and one cysteine-rich domain from

the extracellular domain of the receptor. The N-terminal truncated TrkB receptors can

be phosphorylated even though they are not targeted to the membrane and most are

unable to be activated by BDNF (Luberg et al., 2010).

2.1.6 Functional domains and post-transcriptional processing of TrkB

The extracellular domain of Trk receptors consists of a membrane-targeting signal

peptide, two cysteine clusters that are located around LRRs followed by two

immunoglobulin G (IgG)-like structures adjacent to the transmembrane domain

(Schneider and Schweiger, 1991) (Fig 2B). The intracellular domains of TrkB include a

tyrosine kinase domain and tyrosine motifs. Tyrosine phosphorylation controls the

kinase activity of TrkB receptors and can regulate the binding of adaptor molecules to

the receptor (Segal et al., 1996). The catalytic domain inside the tyrosine kinase domain

is highly conserved among all the receptor tyrosine kinases (Klein et al., 1989; Lee et al.,

2001a; Segal et al., 1996). The IgG-like domain adjacent to the transmembrane domain

acts as a binding site for neurotrophins and determines the ligand specificity of the

receptor (Urfer et al., 1995). Neurotrophin binding to this domain can induce activation

of the catalytic tyrosine kinase domain of the receptor. The other IgG domain and the

leucine- and cysteine-rich repeats also seem to participate in ligand binding either

directly or by inducing conformational changes (Huang and Reichardt, 2003; Ninkina et

al., 1997). In general, the LRRs are thought to participate in protein-protein interactions.

Deletion of the LRRs prevents the ligand from binding to TrkB and blocks the survival

enhancing effects of BDNF in serum-depleted NIH3T3 cells (Ninkina et al., 1997).

Specifically the second LRR of TrkB seems to bind BDNF (Windisch et al., 1995). Co-

expression of p75NTR can alter the extracellular sites of TrkB required for BDNF binding

(Zaccaro et al., 2001). In the absence of p75NTR BDNF binds to the IgG-C2 domain of

TrkB but when p75NTR and TrkB are expressed together, BDNF binding requires the LRR

9

and the cysteine 2 domains. Juxtamembrane domain (short sequence of about 80 amino

acids that is located between the transmembrane and tyrosine kinase domain) of TrkB

regulates the internalization of the receptor after ligand binding (Sommerfeld et al.,

2000). The extracellular domain of TrkB can be N-glycosylated and the glycosylation of

the receptor appears to increase during early development (Fryer et al., 1996; Haniu et

al., 1995). Glycosylation is required for the localization of the Trk receptors to the cell

membrane and can inhibit ligand-independent activation of the receptor (Watson et al.,

1999a)

2.1.7 Expression and subcellular localization of TrkB

TrkB receptor is widely expressed in both the peripheral and central nervous systems. In

situ hybridization analysis has shown that trkB transcripts are expressed in the cerebral

cortex, hippocampus, thalamus, choroid plexus, granular layer of the cerebellum, brain

stem and spinal cord (Klein et al., 1993). In the PNS trkB transcripts have been detected

in the cranial ganglia, retina, ophthalmic nerve, vestibular system, multiple facial

structures, submaxillary glands and dorsal root ganglia. TrkB protein is found in

olfactory bulb, hippocampus, thalamus, hypothalamus, septum, basal ganglia, midbrain

nuclei and cerebellum (Yan et al., 1997).

The expression patterns of the full-length and truncated TrkB receptors differ in the CNS.

During early development TrkB.FL is the main receptor in the brain and is highly

expressed in the dendrites; however, around postnatal day 10-15 (P10-P15) the

expression of the TrkB.T1 receptor increases and exceeds the TrkB.FL especially in

cortical areas (Fryer et al., 1996). In human prefrontal cortex the expression of TrkB.FL

mRNA and protein peaks in toddlers and decreases slightly with aging, whereas the

expression of the TrkB.T1 mRNA is regulated the opposite way with lowest expression

levels in toddlers (Luberg et al., 2010). The TrkB.T1 protein expression seems to increase

until teenage years after which it declines slightly. TrkB.Shc expression is low compared

to the other two isoforms and the expression is reduced during aging compared to the

expression levels in infants (Luberg et al., 2010).

Functional full-length TrkB receptors can be found in postsynaptic densities (Wu et al.,

1996). TrkB receptors are found in axons and dendrites intracellularly and on the cell

surface throughout development (Gomes et al., 2006). During maturation TrkB

receptors localize to excitatory synapses in cortical neurons. The expression pattern of

TrkB in interneurons and their responsiveness to neurotrophins varies during

development (Gorba and Wahle, 1999). The TrkB receptor is not expressed by all types

of interneurons but especially interneurons expressing parvalbumin coexpress TrkB and

BDNF-induced TrkB activation can promote parvalbumin expression via

phosphoinositide 3-kinase (PI3K) signaling in early developmental timepoints (Patz et

al., 2004).

In cultured cells the truncated form of TrkB was heavily expressed on the cell surface in

normal conditions but high levels of the full-length receptor were found in granular

structures near the cytoplasm, suggesting that the majority of TrkB receptors are located

inside the cell (Haapasalo et al., 2002). Similarly to BDNF, the synthesis, expression and

intracellular transport of TrkB is regulated by neuronal activity (Merlio et al., 1993). Du

et al. (2000) found that in cultured neurons TrkB receptors are mainly located in the

cytoplasm and after electrical stimulation are recruited heavily to the membrane.

10

Apparently the receptors can be quickly translocated to the membrane from intracellular

pools e.g. by BDNF stimulation or as a result of increased neuronal activity. In

hippocampal neurons high frequency electrical stimulation induced surface expression

of both the full-length and truncated TrkB receptors in a calcium- and

calcium/calmodulin-dependent protein kinase II (CaMKII)-dependent manner (Du et

al., 2000). Increase in calcium concentration leads to a cyclin-dependent kinase 5 (Cdk5)

-dependent phosphorylation of the receptor at serine 478 which induces TrkB insertion

into the cell membrane (Zhao et al., 2009). In cultured retinal ganglion cells and spinal

motor neurons full-length TrkB receptors were inserted into the cell membrane after an

increase in the amount of intracellular cyclic adenosine monophosphate (cAMP) (Meyer-

Franke et al., 1998). TrkB can also be inserted to the cell membrane after intracellular

transactivation (Puehringer et al., 2013). It has been suggested that TrkB could act as a

“synaptic tag” for plasticity promoting proteins (e.g. BDNF) to promote late-phase long-

term potentiation (L-LTP) since its expression on the plasma membrane increases after

neuronal activity thereby marking active synapses (Lu et al., 2011).

In addition to the insertion of TrkB into the cell membrane, neuronal activity and

increase in intracellular calcium concentration regulate the internalization of TrkB

receptors (Du et al., 2003). The kinetics of the receptor insertion to the membrane and

its subsequent internalization can define the signaling pathways and other downstream

actions of the receptor. A short treatment (15 s) with potassium chloride (KCl) did not

increase TrkB surface expression in hippocampal primary neurons or TrkB.TK+

transfected N2a cells but BDNF did (Haapasalo et al., 2002). However, a 5 minute

treatment with BDNF already reduced the surface expression of TrkB and the levels

remained low for at least 24h, possibly due to endocytosis following receptor activation.

Pretreatment of neuronal cell cultures with KCl prevented the decrease in TrkB surface

expression that normally occurs after BDNF stimulation elongating the effect of BDNF

on TrkB signaling, including phosphorylation of the receptor and its downstream targets

extracellular signal-regulated kinase (ERK), protein kinase B (Akt) and PLCγ (Guo et al.,

2014). Inserting more TrkB receptors to the cell membrane compensates for the

endocytosis occurring after ligand binding, and the TrkB signaling shifts from a transient

event to sustained state.

After endocytosis receptors are brought back to the membrane, degraded or transported

towards the cell soma depending on whether the receptors are targeted to recycling

endosomes, early endosomes or late-endosomes/lysosomes (IJzendoorn, 2006).

TrkB.T1 and TrkB.FL receptors seem to be differentially recycled after BDNF-induced

endocytosis with TrkB.FL receptor degraded (targeted to the lysosomes) more quickly

than TrkB.T1 (Huang et al., 2009). The Rab11-positive endosomes regulate dendritic

trafficking of the TrkB receptors after ligand binding and have an important role in the

dendritic branching promoting effects of BDNF (Lazo et al., 2013). The Rab11-positive

endosomes carrying TrkB receptor enrich to dendrites and increase TrkB expression in

the plasma membrane (Watson et al., 1999b).

Recently, cell-surface protein SLIT- and NTRK-like protein 5 (Slitrk5) has been

implicated in the regulation of BDNF-induced TrkB signaling (Song et al., 2015). The

extracellular LRR domain 1 of Slitrk5 specifically interacts with the LRR-domain of TrkB

receptors after BDNF stimulation. In the absence of Slitrk5 BDNF stimulation induced

normal TrkB phosphorylation, however, prolonged BDNF treatment did not produce

11

increase in primary dendrite formation as was seen in wild type neuronal cultures. The

authors found that TrkB degradation was increased in the absence of Slitrk5 due to

reduced targeting of TrkB to recycling endosomes, which are responsible for the

recycling of the endocytosed receptors back to the membrane. These findings implicate

Slitrk5 in targeting of TrkB to recycling endosomes and regulation of TrkB signaling.

Receptor internalization and the following dynein-dependent transport of TrkB receptor

from axon to soma (retrograde transport) are necessary for its survival-promoting effects

in sensory neurons (Heerssen et al., 2004). The first observation of retrograde transport

of activated Trk receptors was done using sciatic nerve injury, after which the

phosphorylated Trk receptors were accumulating in the distal side of the injury

indicating that the receptors were transported in clathrin-coated vesicles from the axon

terminal towards the soma (Bhattacharyya et al., 1997). In addition, anterograde

transport of TrkB following sciatic nerve injury has been reported (Yano et al., 2001).

2.1.8 TrkB activation by BDNF and downstream signaling

TrkB is a receptor tyrosine kinase and thus catalyzes upon activation transfer of a

phosphate group to a tyrosine of another protein. TrkB receptors form homodimers upon

ligand binding and this allows the receptors to phosphorylate tyrosines 706 and 707 in

each other’s catalytic domain resulting in increased kinase activity of the receptor

(Reichardt, 2006). In addition to the catalytic domain, other tyrosines of the receptor

can be phosphorylated with the most extensively studied phosphorylation sites being

Y515 and Y816 (Middlemas et al., 1994; Segal et al., 1996). Phosphorylated Y515 and

Y816 (pY515 and pY816, respectively) can serve as docking site for Src homology 2 (SH2)

adaptor proteins and phosphotyrosine binding domain containing proteins. Shc, Frs2

(fibroblast growth factor receptor substrate 2) and PLCγ are the major interactor

proteins directly binding to TrkB receptors and activating Trk-associated signaling

pathways Ras, PI3k and PLCγ1 (Obermeier et al., 1993) (Fig 3).

Shc binding to pY515 can activate signaling via PI3K and Ras by inducing a cascade of

protein-protein interactions that further recruit serine/threonine kinases Akt and ERK

(Hallberg et al., 1998; Obermeier et al., 1994). ERK can also be activated by signaling

initiated by PLCγ binding to pY816 in TrkB (Stephens et al., 1994). Shc binding site

signaling is linked to survival and axon outgrowth (Atwal et al., 2000). The Shc-PI3K-

Akt pathway and the Shc-Ras-Raf-ERK pathway both promote survival and

differentiation (Yao and Cooper, 1995). In addition to Shc also Frs2 can bind to pY515.

Frs2 interacts with Shp2 and Grb2 to induce ERK activation via Ras (Easton et al., 2006).

It has been suggested that competition of Shc and Frs2 for the binding site could regulate

the induction of proliferation vs. differentiation by the neurotrophins (Meakin et al.,

1999). The survival promoting effects of ERK are mediated via inhibition of pro-

apoptotic factors and increases in transcription of pro-survival factors (Bonni et al.,

1999). BDNF-TrkB-ERK signaling promotes dendritic growth and increases the number

of spines in a subgroup of hippocampal neurons (Alonso et al., 2004). The PI3K-Akt

pathway leads to activation of mTOR which regulates P70S6k and 4eBP1 to promote

translation of proteins that affect cell survival, proliferation, differentiation and dendritic

growth (Kumar et al., 2005; Takei et al., 2004).

In addition to activating ERK, BDNF-TrkB signaling also induces the translocation of

ERK to the nucleus and thus affects the transcription factors regulated by ERK, (e.g.

12

cyclic AMP response element-binding protein, CREB) (Patterson et al., 2001; Ying et al.,

2002). ERK cannot, however, directly phosphorylate CREB but requires Rsk to

phosphorylate the serine 133 in CREB (Watson et al., 2001). CREB is one of the

transcription factors linked to increased transcription of genes required for late-phase

LTP and cell survival (Minichiello et al., 2002; Watson et al., 2001).

Minichiello et al. generated a genetically modified mouse with a point mutation at the

Shc binding site of TrkB receptor (Y515 → phenylalanine (F)) (Minichiello et al., 1998).

Cultured nodose and trigeminal ganglion neurons from these mice responded poorly to

NT4 stimulation, suggesting that the Shc site activation is important for NT4 induced

TrkB activation. In vivo, neurons known to depend on NT4 signaling were missing from

TrkBY515F mice, however, most of the neurons that are lost in BDNF knockout animals

were not affected in the mice with the Shc site mutation. ERK signaling is significantly

reduced in TrkBY515F mice but CREB activation by BDNF stimulation appears to be

normal (Minichiello et al., 1998, 2002). In contrast to the findings in cultured neurons

the TrkBY515F mice did not show any deficits in the differentiation of CNS neurons,

however, a mutation in the Shc site impaired axonal regeneration in vivo (Hollis et al.,

2009; Minichiello et al., 1998).

The PLCγ1 binding site (Y816) of TrkB is in close proximity to the C-terminal region of

the receptor and can be phosphorylated by ligand binding (Middlemas et al., 1994). Upon

binding to pY816 the membrane-bound enzyme PLCγ is activated and can then hydrolyze

phosphatidyl(4,5)inositolbisphosphate (PIP2) to second messengers diacylglycerol

(DAG) and IP3 (Carpenter and Ji, 1999). DAG is a lipid that cannot diffuse into the

cytoplasm but stays in the plasma membrane and activates protein kinase C (PKC)

signaling. In contrast, IP3 can enter the cytoplasm and activate the release of calcium

from the intracellular storages. PKC activation and calcium release can lead to the

activation of ERK, CaMKIV, and CREB and to release of neurotrophins (Canossa et al.,

2001; Finkbeiner et al., 1997; West et al., 2001). BDNF can potentiate excitatory synaptic

transmission by increasing intracellular calcium concentration via TrkB-PLCγ-IP3-PKC

signaling (Carmignoto et al., 1997; Levine et al., 1995; Li et al., 1998). In contrast to Y515,

Y816-mediated signaling seems to be required for synaptic plasticity, especially for

hippocampal long-term potentiation (Korte et al., 2000; Minichiello et al., 2002).

TrkB-mediated PLCγ activation is also required for epileptogenesis (Gu et al., 2015; He

et al., 2010). TrkB Y816 phosphorylation is increased during status epilepticus and

preventing the signaling of the Y816-residue reduced PLCγ activation and prevented the

epileptogenesis in a kindling model (He et al., 2010). Instead of blocking the TrkB

receptor, inhibiting just the coupling of PLCγ to pY816 after chemically induced seizures

prevents the epileptogenesis but does not impair the TrkB-mediated promotion of

survival (Gu et al., 2015).

In the PNS neurotrophin-induced activation of TrkB receptors in the nerve terminal

results in endocytosis of the receptor-ligand complex and retrograde transport of the

complex to the soma resulting in CREB activation and induction of immediate-early gene

c-fos (Watson et al., 1999b). The retrograde transport of activated Trk receptors recruits

specific signaling pathways involving ERK5 to mediate the survival promoting effects in

the soma, suggesting that the location of TrkB activation also controls the downstream

signaling pathways (Watson et al., 2001). Clathrin- and dynamin-dependent endocytosis

13

of TrkB receptors after BDNF stimulation has been shown to be important for BDNF-

induced PI3K-Akt signaling (Zheng et al., 2008).

Fig 3. The main signaling pathways of the TrkB neurotrophin receptor. The TrkB receptor can be phosphorylated upon BDNF binding to the catalytic domain Y706/7, the Shc binding site Y515, and the PLCγ1 binding site Y816. The main pathways include Shc-PI3K-Akt, Shc-PI3K-Ras-Raf-ERK and PLCγ1-IP3–PKC-CaMKIV that promote survival, differentiation, calcium release and initiation of transcription and translation.

14

2.1.9 Signaling of the truncated TrkB receptor

Alternative splicing of the TrkB transcript results in the truncated receptors TrkB.T1 and

TrkB.T2 that lack the intracellular tyrosine kinase domain but have small intracellular

domains of 23 and 21 amino acids, respectively (Baxter et al., 1997; Klein et al., 1990).

The truncated TrkB receptors can bind and internalize BDNF, however, due to the lack

of tyrosine kinase domain the canonical neurotrophin signaling responses cannot be

activated. BDNF stimulation of Xenopus oocytes transfected with TrkB.FL, TrkB.T1 or

TrkB.T2 increased calcium efflux in a phosphatidylinositol dependent manner only in

TrkB.FL transfected cells indicating that BDNF is able to activate this signaling only via

tyrosine kinase containing TrkB receptors (Eide et al., 1996).

When discovered, the truncated Trk receptors were thought to act as “simple” scavengers

which limit the diffusion of neurotrophins since they are expressed widely in non-

neuronal tissues and can internalize BDNF after binding (Biffo et al., 1995). However,

when the truncated receptor is expressed together with the full-length receptor it can

form a heterodimer and inhibit the tyrosine kinase activity of TrkB.FL in a dominant-

negative manner (Eide et al., 1996; Haapasalo et al., 2001). The truncated receptors can

reduce the surface expression of the full-length TrkB and thus regulate the availability of

TrkB receptors to its ligands (Haapasalo et al., 2002). Because of their dominant negative

function truncated receptors can negatively affect the survival role of BDNF (Ninkina et

al., 1996). In vivo deletion of truncated TrkB receptors could partially rescue the

phenotype of BDNF heterozygous knockout mice suggesting that the truncated TrkB

receptor negatively regulates the full-length TrkB signaling when expressed at

physiological levels (Carim-Todd et al., 2009). Moreover, overexpression of TrkB.T1

reduced TrkB phosphorylation in vivo (Saarelainen et al., 2000).

The effects of the truncated TrkB receptor are not limited to the regulation of the TrkB.FL

signaling; they can also initiate intracellular signaling themselves. The small intracellular

domains of the truncated receptors are required for this signaling (Baxter et al., 1997).

BDNF stimulation can induce release of calcium from intracellular storages through

TrkB.T1-activated IP3 signaling in the absence of TrkB.FL (Rose et al., 2003). More

precisely, Rho GDI1 can bind the intracellular domain of TrkB.T1 and upon ligand

binding dissociates from TrkB.T1 and activates other Rho GTPases that initiate changes

in astrocytic function (Fenner, 2012).

2.1.10 TrkB transactivation

Receptor transactivation occurs independently of the ligand binding via activation of

intracellular signaling events. Studies concerning the transactivation of Trk receptors

have been mostly done in vitro and it is yet to be confirmed whether the same

phenomena exists in physiological conditions in vivo. The first demonstrations of Trk

receptor transactivation came from studies done in PC12 cells and primary hippocampal

neurons, where G-protein coupled receptor (GPCR) ligands adenosine and pituitary

adenylate cyclase-activating polypeptide (PACAP) were shown to activate Trk receptors

without direct binding to the receptors or effects mediated via neurotrophins (Lee and

Chao, 2001; Lee et al., 2002). The activation was mediated by G-protein coupled

adenosine A2 or PAC1 receptors and occurred relatively slowly, requiring a minimum of

90 minutes. Adenosine and PACAP can also activate the PI3k-Akt-pathway via Trk

receptors and to mediate cell survival. The effects of transactivation on ERK

15

phosphorylation are somewhat controversial since PACAP stimulation resulted in a

sustained phosphorylation of ERK whereas adenosine stimulation did not. Intracellular

calcium chelator EGTA and protein phosphatase 1 (PP1) (and PP2, Rajagopal and Chao

2006) blocked the effects, suggesting that intracellular calcium and Src family members

could be involved in the transactivation mechanism. The specific member of the Src

family kinases involved in Trk transactivation is suggested to be Fyn (Rajagopal and

Chao, 2006). Phosphorylation of Fyn is seen with similar temporal pattern as Trk

activation, and it also requires increases in intracellular calcium concentration and can

be blocked by PP1. Fyn was also shown to interact with Trk receptor juxtamembrane

region, but this interaction is not dependent on Trk kinase activity. Also inhibiting

transcription and translation with actinomycin D and cycloheximide, respectively,

abolished the effect of GPCR ligands on Trk phosphorylation (Rajagopal et al., 2004).

The ability of adenosine A2A receptor agonist to transactivate TrkB receptors was later

demonstrated in vivo and the TrkB transactivation was crucial for the survival promoting

effects of A2A agonist (Wiese et al., 2007).

In addition to GPCR ligands, zinc has been reported to activate TrkB receptors and its

downstream signaling pathways in a BDNF independent manner (Huang et al., 2008).

In contrast to GPCR-mediated activation, the zinc-induced TrkB activation occurs

quickly (in 5 minutes), thus, with similar kinetics as BDNF-induced TrkB activation.

Interestingly, also reactive oxygen species (ROS) can activate TrkB receptor via an

intracellular mechanism requiring zinc (Huang and McNamara, 2012). Zinc can enter

the cell via the ionotropic NMDA-receptors and activate the Src family kinases similarly

to other transactivators. Zinc was also shown to increase LTP of mossy fiber-CA3

synapses via transactivation of TrkB receptors. However, later a study from the same

group failed to show that vesicular zinc is required for basal TrkB activation in adult

mouse brain (Helgager et al., 2014). There is some controversy surrounding the role of

zinc to transactivate TrkB receptors in cell culture, because it has been suggested that

TrkB activation by zinc requires rapid processing of proBDNF to BDNF, suggesting that

TrkB activation is mediated by matrix metalloproteinase -facilitated BDNF cleavage and

not by transactivation (Hwang et al., 2005).

In addition to GPCR ligands and zinc, low-density lipoprotein receptor-related protein 1

(LRP1) agonists transactivate TrkB receptors in PC12 cells quickly (10 minutes) (Shi et

al., 2009). Src family kinases were required also for the LRP1-induction of TrkB

activation.

During embryonic development, TrkB transactivation has been shown to regulate the

migration of cortical precursor cells and developing interneurons (Berghuis et al., 2005;

Puehringer et al., 2013). At an early embryonic stage (E11) TrkB receptors in cortical

precursor cells do not respond to BDNF stimulation but instead are activated by

epidermal growth factor (EGF) (Puehringer et al., 2013). At this developmental

timepoint the TrkB receptors are located mainly intracellularly and cannot be activated

by BDNF. However, EGF can transactivate an intracellular pool of Trk receptors that can

then subsequently be inserted to the cell membrane. Trk receptor transactivation by EGF

is required for the proper migration of the cortical precursor cells, but does not affect the

survival of the cells (Puehringer et al., 2013). TrkB also regulates the migration of

interneurons via transactivation by endocannabinoid anandamide (Berghuis et al.,

2005). TrkB transactivation by EGF and anandamide occurs much faster than the

16

adenosine or PACAP induced transactivation, but Src family kinases are essential for the

transactivation in all the experiments.

Interestingly, transactivation of Trk receptors seems to induce a strong phosphorylation

of a different form of Trk receptors of 110 kDa in size in addition to the normal 145 kDa

protein and the regulation of this protein can occur even faster than that of the full-length

form (Lee and Chao, 2001; Lee et al., 2002; Rajagopal et al., 2004). The transactivation

has been shown to preferentially activate intracellular Trk receptors (Rajagopal et al.,

2004). Because transactivation occurs intracellularly, immaturely glycosylated Trk

receptors in trans-golgi can be phosphorylated to produce the phosphorylated 110 kDa

protein (Rajagopal et al., 2004; Schecterson and Bothwell, 2010). In fact, treatment with

Brefeldin A, an inhibitor of protein transport from endoplasmic reticulum to Golgi

apparatus, increased the amount of 110 kDa protein (Rajagopal et al., 2004).

Interestingly PACAP-induced TrkB transactivation resulted in reversible Golgi

fragmentation (Schecterson et al., 2010). It is not clear what the consequences of the

Golgi fragmentation induced by TrkB transactivation are, however, it is hypothesized

that it could lead to redistribution of Golgi fragments to the dendrites and affect post-

translational processing of dendrite targeted mRNAs. On the other hand, Golgi

fragmentation can play a role in the pathophysiology of neurodegenerative disorders,

thus suggesting that it may be harmful (Schecterson et al., 2010).

It has been suggested that TrkB receptors are expressed as dimers intracellularly before

ligand binding, which could facilitate transactivation (Shen and Maruyama, 2012). In

addition to transactivation ligand independent TrkB activation can be experimentally

achieved with light stimulation using genetically modified TrkB receptors that have a

light-activatable domain inserted into the intracellular domain (Chang et al., 2014).

Light-induced TrkB dimerization and activation led to activation of the ERK signaling

and increase in neurite outgrowth.

2.1.11 ProBDNF and p75NTR signaling

In addition to Trk receptors all neurotrophins bind to and activate the p75NTR

(Rodriguez-Tébar et al., 1990, 1992). As discussed in preceding chapters these two

receptors can have synergistic, distinct or even opposite effects on neuronal function.

The p75NTR receptor belongs to the tumor necrosis factor receptor family and can interact

with multiple co-receptors and depending on the interaction partner initiate signaling

via different signaling pathways (Dechant and Barde, 2002). P75NTR has been shown to

increase the specificity and the sensitivity of the Trk receptors to neurotrophins (Bibel et

al., 1999; Davies et al., 1993). Upon neurotrophin binding p75NTR can inactivate RhoA

resulting in neurite growth. Some factors, such as NOGO, however, can promote the

interaction between p75NTR and Rho-GDI leading to increased RhoA activity and

inhibition of neurite growth (Yamashita and Tohyama, 2003). Moreover, neurotrophin

binding to p75NTR in the absence of the Trk receptors can induce neuronal death via

activation of JUN kinase (Bamji et al., 1998; Friedman, 2000).

Most importantly, proneurotrophins bind to p75NTR with much higher affinity than

mature neurotrophins (Lee et al., 2001b). Indeed, proBDNF, but not mature BDNF, can

activate apoptotic signaling in the neurons via binding to a preformed complex of p75NTR

and sortilin (Nykjaer et al., 2004; Teng et al., 2005). Activation of survival signaling via

Trk receptor, however, can rescue the cells from the apoptosis in a competitive manner.

17

Thus, pro and mature neurotrophins are able to elicit completely opposing functions and

the cleavage of neurotrophins importantly determines the effects that follow the release

of neurotrophins. Naturally, the signaling effects of proneurotrophins require that they

have to be secreted and not cleaved extracellularly. However, it is still not certain to

which extent proBDNF is released in vivo.

In Xenopus nerve-muscle co-cultures the activity-dependent increase in the cleavage of

proBDNF to BDNF supports the establishment of the active synapse via TrkB and p75NTR,

while in the non-active synapse proBDNF stimulates the retraction of the synaptic

terminal (Je et al., 2012). Similar results were obtained from in vivo experiments using

mouse neuromuscular junctions, namely that proBDNF promotes the synapse

elimination through p75NTR and sortilin and mature BDNF supports the survival of the

synapses via TrkB (Je et al., 2013). Synaptic depression is important in the excitatory

neuron synaptic clustering, which is considered to be involved in the regulation of

computational power of the neuron. ProBDNF signaling via the p75NTR mediates the

decreased transmission of synapses that are active in an asynchronous manner

compared to their neighboring synapses (Winnubst et al., 2015).

The role of proBDNF cleavage to BDNF has been studied also in electrophysiological

experiments. Pang et al. (2004) demonstrated that the cleavage of proBDNF to BDNF

after secretion is required for the late phase LTP (L-LTP) in hippocampal slices. The

levels of hippocampal proBDNF in tPA or plasminogen knock out mice are increased

indicating that these enzymes are involved in the processing of proBDNF. Indeed, tPA

and plasminogen deficient mice show impaired LTP that can be rescued by application

of exogenous BDNF (Pang et al. 2004). The cleavage of proBDNF by plasmin is

important for the LTP since cleavage resistant proBDNF cannot rescue LTP in

plasminogen or tPA deficient mice. ProBDNF itself had no effect on LTP, however, it

enhanced LTD. Later it was shown that the facilitation of the LTD by proBDNF depends

on the p75NTR (Woo et al., 2005). High frequency stimulation (HFS) is required for LTP

and in hippocampal neuronal cultures it has been shown that HFS results in release of

tPA together with BDNF/proBDNF (Nagappan et al., 2009). In the presence of tPA more

mBDNF is formed, which is important for the L-LTP. In contrast low frequency

stimulation (required for LTD) increases the extracellular ratio of proBDNF to BDNF

since it does not increase secretion of tPA. Thus, the extracellular cleavage of proBDNF

to BDNF determines also postsynaptic effects of BDNF.

In BDNF conditional knockout mice the LTD was not affected in hippocampal slices (but

LTP was impaired), suggesting that proBDNF is not necessary for LTD (Matsumoto et

al. 2008). In p75NTR knockout mice, however, especially the NMDA receptor-dependent

LTD was impaired (Woo et al., 2005). Also mice expressing proconvertase/furin

cleavage-resistant proBDNF have decreased dendritic complexity in the hippocampus,

impaired theta burst stimulation-induced LTP and enhanced LTD in Schaffer collaterals

(Yang et al., 2014).

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2.2 Role of BDNF and TrkB in CNS development and function

2.2.1 Cell survival and differentiation

The neurotrophins are essential for the development of the nervous system, and are

known to regulate physiological functions also in the adulthood. During development

neurotrophins are required to support the survival of a limited number of neurons that

compete for target innervation. This applies mostly to the PNS and has been extensively

studied in relation to survival of peripheral neurons. The applicability of the

neurotrophin hypothesis to the CNS has not been reliably proven but according to

current data it does appear to apply to certain populations of neurons. Notably,

significant proportion of CNS neurons also undergoes apoptosis during early

development. An interesting finding suffests that in contrast to TrkA and TrkC receptors

TrkB receptor does not seem to be a dependence receptor that would induce apoptosis

in the absence of ligand (Nikoletopoulou et al., 2010).

In general, neurons of the PNS are more dependent on certain neurotrophin and/or

certain neurotrophin receptor compared to neurons in the CNS. Mice with complete

deletion of BDNF or TrkB do not survive after birth because of deficits in peripheral

nervous system innervation (Ernfors et al., 1994; Klein et al., 1993). Specific deficits in

the nodose-petrosal ganglion, which brings gastrointestinal, cardiac and respiratory

information to the CNS, results in problems with breathing and cardiac function that

may underlie the fatal phenotype of the BDNF and TrkB knockout mice (Conover et al.,

1995; Erickson et al., 1996; Jones et al., 1994; Silos-Santiago et al., 1997). TrkB knockout

mice also have reduced amounts of vestibular neurons and loss of semicircular canal

innervation causing defects in balance. TrkB or BDNF deletion does not cause gross

deficits in the CNS, but decreases the amount of interneuron markers suggesting that

BDNF-TrkB signaling is specifically involved in interneuron maturation and function

(Alcántara et al., 1997). Parvalbumin expression is delayed in the TrkB knockout mouse

hippocampus and cortex and cell death is increased during the first postnatal weeks

compared to wild type mice in the cortex, hippocampus (especially dentate gyrus),

striatum, septum, thalamus and olfactory bulb with specific temporal patterns (striatum

earlier, dentate gyrus later) (Alcántara et al., 1997; Minichiello and Klein, 1996). Proper

parvalbumin expression in interneurons and formation of GABAergic inhibitory

synapses requires BDNF, TrkB and neuronal activity (Patz et al., 2004; Rico et al., 2002).

BDNF and TrkB are involved in the regulation of dendritic branching, adjusting the

number of dendritic spines and in synapse formation for example in some neuronal

populations of the hippocampus and cortical layer 4 (Luikart et al., 2005; Martınez et al.,

1998; McAllister et al., 1995; Xu et al., 2000a). In vivo some striatal neurons require

BDNF and TrkB for survival during development and for dendritic complexity in

adulthood (Baydyuk et al., 2011; Rauskolb et al., 2010). Also migration of neurons in the

developing neocortex is delayed in mice lacking TrkB receptor affecting the stratification

of the cortex and differentiation of neurons (Medina et al., 2004).

The effects of BDNF-TrkB signaling on the differentiation and survival of neurons have

been mainly studied in vitro. BDNF increases the differentiation of neuronal precursor

cells and promotes neurite outgrowth and survival of cultured neurons (Ahmed et al.,

1995; Baj et al., 2011; Kirschenbaum and Goldman, 1995; Vicario-Abejón et al., 1998).

BDNF stimulation also increases the number of excitatory and inhibitory synapses in

19

cultured hippocampal neurons and seems to affect especially the neurite outgrowth of

GABAergic cells supporting the in vivo findings that BDNF is required for the normal

development of inhibitory interneurons. Reduction in markers for GABAergic synapses

could also be found when TrkB kinase function was inhibited for 20 days suggesting that

the maintenance of the GABAergic synapses requires TrkB signaling also during

adulthood (Chen et al., 2011). Signaling via the PLCγ binding site of the TrkB receptor

seems to be important for these effects.

2.2.2 Plasticity

The interest to the possible plasticity promoting effects of neurotrophins were raised by

the findings that seizure activity and sensory stimulation led to increased expression of

neurotrophins (Thoenen, 1995). Neuronal plasticity is the ability of the brain to change

in response to experience, environment and intrinsic activity. The processes regulating

brain plasticity on a molecular or structural level involve the strengthening or weakening

of synapses and the formation or elimination of synaptic connections. Experimental

approaches to elucidate plasticity include analysis of neurogenesis, dendritic spines and

long-term potentiation.

Neurogenesis

Formation of new neurons and their integration into existing neural networks is an

important mechanism of neuronal plasticity. Neurogenesis in the adult brain was a

matter of debate for decades but now it is well accepted that adult neurogenesis takes

place in the subventricular zone (SVZ) of lateral ventricles and subgranular zone (SGZ)

of dentate gyrus (Zhao et al., 2008). Mice that lack BDNF have increased apoptosis

around the subventricular zone and in the dentate gyrus after the first two postnatal

weeks (Linnarsson et al., 2000). Voluntary exercise on a running wheel or an enriched

environment are well-known means to enhance neurogenesis in laboratory animals

(Zhao et al., 2008). The main factors that are known to negatively affect neurogenesis

are stress (glucocorticoids) and aging.

In dentate gyrus SGZ there are neural progenitor cells that continue to divide also in the

adult brain producing neurons that migrate to the granule cell layer and extend dendrites

to the molecular layer and axons to the CA3 region (van Praag et al., 2002). The basal

level of bromodeoxyuridine (BrdU) -labeled adult-born neurons is normal in BDNF

conditional knockout mice (BrdU incorporates into newly synthesized DNA), however,

following environmental enrichment and voluntary exercise the amount of BrdU labeled

cells increased more in the hippocampus of wild type mice compared to BDNF

conditional knockout mice (Choi et al., 2009). BDNF deletion affected the survival of

adult-born neurons, resulting in significantly reduced levels of BrdU-labeled cells

measured 4 weeks after the BrdU injection when the mice were housed in a standard or

enriched environmental conditions. BDNF heterozygous knockout mice also show

reduced survival of hippocampal new-born neurons after environmental enrichment or

chronic antidepressant treatment (Rossi et al., 2006; Sairanen et al., 2005).

Intrahippocampal infusion of BDNF increased the amount of new-born neurons, but

unexpectedly more in the contralateral side of the infusion, suggesting that BDNF does

not directly affect these cells since BDNF presumably does not diffuse widely from the

injection site (Scharfman et al., 2005).

20

Type B and ependymal cells in the SVZ express high levels of TrkB and low levels of

BDNF (Galvão et al., 2008). The TrkB receptor expressed in SVZ is, however, mainly the

truncated form of the receptor (TrkB.T1). TrkB expression co-localized with BrdU

suggesting that stem cells of the SVZ express TrkB (Galvão et al., 2008). When the stem

cells from TrkB knockout mice were grafted to adult wild type mice they did not show

any difference when compared to grafts from wild type mice in regards to their ability to

give rise to new neurons or in the migration or differentiation of the olfactory bulb

neurons; suggesting that TrkB-mediated signaling is not critical for these effects (Galvão

et al., 2008). However, similarly to hippocampus, TrkB signaling may be necessary for

the maturation or survival of the cells in SVZ.

Intraventricular administration of BDNF has been reported to increase the amount of

BrdU labeled olfactory bulb neurons in the adult rat brain (Benraiss et al., 2001; Zigova

et al., 1998). However, an osmotic minipump releasing BDNF for 14 days to the lateral

ventricle had no effect on the amount of new cells in the olfactory bulb of mice and, in

fact, decreased the BrdU-labeled cells in the olfactory bulb of rats likely due to decreased

proliferation of SVZ cells (Galvão et al., 2008). Infection of anterior SVZ cells with

BDNF-expressing virus increased proliferation and/or survival of neural progenitor cells

(Henry et al., 2007). Also in both of these studies increased BrdU-labeling was noticed

in the striatum. Striatal neurogenesis has not been fully observed in the adult brain, yet,

from human studies there is some evidence that new neurons can be found in adult

striatum (Ernst et al., 2014). Damage to some brain areas can result in increased

neurogenesis, and BDNF has been shown to have controversial effects in these situations.

In a study by Henry et al. (2007) BDNF increased the amount of BrdU positive cells in

chemically lesioned striatum. In another study, however, long-term hippocampal viral

expression of BDNF in dentate gyrus prevented the formation of new-born after ischemic

injury (Larsson et al., 2002).

Long-term potentiation

Long-term potentiation, LTP, is a persistent increase in the synaptic strength induced by

brief high frequency electrical stimulation or coincident activation of pre- and

postsynaptic neurons (Minichiello, 2009). LTP is considered as a cellular mechanism

underlying learning and memory.

Application of BDNF directly to hippocampal slices enhances synaptic strength in the

Schaffer collateral-CA1 synapse in a TrkB-dependent manner (Kang and Schuman,

1995). In contrast, LTP in Schaffer collaterals is impaired in BDNF heterozygous

knockout mice (Korte et al., 1995). However, BDNF knockout and BDNF heterozygous

knockout mice had similar impairments in LTP, with LTP being induced in some slices

also in the absence of BDNF, indicating that in all conditions normal levels of BDNF are

not necessary for LTP. Viral expression of BDNF in CA1 neurons or application of

recombinant BDNF can mostly rescue impaired LTP, suggesting that BDNF itself is

required for LTP and the deficits are not caused by abnormalities due to the lack of BDNF

during the development (Korte et al., 1995; Patterson et al., 1996). The late-phase LTP in

particular seemed to be affected in the absence of BDNF (Korte et al., 1996). Activation

of presynaptic NMDA receptors by neuronal activity results in increased intracellular

calcium concentration that triggers the release of BDNF from presynaptic terminals

(Park et al., 2014). BDNF is released from the presynaptic neurons presumably together

with glutamate to induce LTP and in this manner seems to specifically potentiate the

21

highly active synapses (Gottschalk et al., 1998; Zakharenko et al., 2003). This is further

supported by the finding that application of BDNF to dendrites, but not to axons, evoked

a calcium response and induced LTP when combined with brief electrical stimulation

(Kovalchuk et al., 2002). Long-term spine enlargement related to LTP requires that

BDNF is secreted specifically in the active synapses (Tanaka et al., 2008). BDNF

promotes spine enlargement via activation of cofilin and p21 activated kinase that are

involved in regulation of spine morphogenesis (Rex et al., 2007).

The effects of BDNF on LTP are mediated through TrkB receptor and requires especially

the signaling initiated by PLCγ binding to Y816 of TrkB (Minichiello et al., 2002). In

addition to experiments using slices, the impairment of LTP in mice with point mutation

in TrkB PLCγ binding site was also demonstrated by in vivo electrophysiological

recordings (Gruart et al., 2007). It has also been suggested that BDNF-induced activation

of presynaptic TrkB receptors is required for LTP (Xu et al., 2000b). To support the

requirement for both presynaptic and postsynaptic TrkB, intact presynaptic and

postsynaptic PLCγ signaling is required for hippocampal LTP (Gärtner et al., 2006).

Interestingly, mice overexpressing TrkB show increased PLCγ activation and enhanced

spatial learning but impaired LTP (Koponen et al., 2004). It was suggested that the

baseline synaptic activity in these mice is enhanced, which could lead to occlusion of LTP.

BDNF-TrkB in visual cortex plasticity

Basic organization of the brain occurs during early development independently of

external stimuli. Molecular factors defined by genetics and intrinsic activity in the brain

result in the formation of basic neuronal networks (Levelt and Hübener, 2012). A critical

period is a well-defined time window during which environmental sensory stimulus is

required for proper wiring and fine-tuning of neuronal networks in the brain. During the

critical period the sensory stimulus, or lack there of, permanently affects the network in

question. Sensory stimuli have less impact on the neuronal wiring before and after the

critical period. In addition to sensory systems, it has been suggested that critical period

exists in fear extinction (Gogolla et al., 2009). The molecular mechanisms of critical

periods have been extensively studied and understanding how to enhance plasticity of

the adult brain could be beneficial in the treatment of psychiatric disorders, which often

have a developmental origin possibly caused by abnormal critical period and are difficult

to treat when symptoms appear in adulthood.

Visual cortex is a generally-used model for studying critical period plasticity in the rodent

brain. During the critical period of binocular vision formation the local inhibitory

neurons in the primary visual cortex mature (Hensch, 2005). During the critical period

it is possible to permanently alter the physical microstructure and organization of the

visual cortex that may result in amblyopia. Apparently the columnar organization of the

ocular dominance columns is well-established before the critical period but the sensory

nformation during the critical period is able to modify the size of the columns and

individualize the ocular dominance maps (Hensch, 2005).

Axons projecting from the lateral geniculate nucleus of the thalamus form eye-specific

ocular dominance columns in the layer IV of the visual cortex during development

(Cabelli et al., 1995). BDNF expression is not necessary for the formation of the eye-

specific columns (Lyckman et al., 2005), however, infusion of TrkB-IgG, which binds to

BDNF and possible other TrkB-binding molecules, can block the formation of the ocular

22

dominance columns suggesting that BDNF together with other endogenous TrkB ligands

mediate this effect (Cabelli et al., 1997). Interestingly, BDNF infusion to the visual cortex

can prevent the ocular dominance column formation by possibly disturbing activity-

dependent competition of target innervation.

Visual input and light exposure regulates bdnf mRNA expression in the visual cortex

(Castrén et al., 1992). If adult rats are kept in total darkness for one week bdnf mRNA

expression in the visual cortex is reduced but can be restored quickly by one hour

exposure to light. Also the normally occurring reduction in the sizes of receptive fields

and improvement of visual acuity is impaired if rats are reared in darkness from birth

(Fagiolini et al., 1994). In transgenic mice overexpressing BDNF in CaMKII-positive

neurons the reduction in visual acuity by dark rearing is prevented (Gianfranceschi et al.,

2003). Interestingly, visual experience is not required for visual acuity in these mice.

Also, the receptive fields of dark-reared BDNF-overexpressing mice were not increased

similarly to dark-reared wild type mice. Dark rearing in wild type mice prolonged the

critical period so that monocular deprivation at P40 still produced a shift in ocular

dominance. In BDNF-overexpressing mice the critical period for visual cortex occurs and

closes earlier in development than in wild type mice and it cannot be delayed by dark

rearing (Gianfranceschi et al., 2003; Hanover et al., 1999; Huang et al., 1999). BDNF-

overexpressing mice show faster maturation of inhibitory interneurons, e.g. parvalbumin

neurons, in the visual cortex (Huang et al., 1999). In contrast, dark rearing delays the

emergence of GABAergic inhibition in the visual cortex in wild type mice, whereas BDNF

overexpression allows for normal emergence of the GABAergic inhibition in the dark-

reared animals (Gianfranceschi et al., 2003). Reduced BDNF levels do not seem to affect

the critical period of the visual system, since BDNF heterozygous knockout mice have

normal critical period (Bartoletti et al., 2002).

2.2.3 BDNF and TrkB mutations and polymorphisms in humans

Information about the role of BDNF and TrkB in humans has been obtained from

patients with genetic mutations in the bdnf and trkb genes. Patients with a rare genetic

disorder, WAGR syndrome, have a chromosomal deletion that in some cases includes the

whole bdnf gene. Patients with heterozygous bdnf deletion show hyperphagia (increased

appetite) and impaired pain sensation and have a higher body mass index (BMI) than

WAGR patients without bdnf deletion (Han et al., 2008). In the patients with bdnf

deletion also the serum BDNF levels were about 50 % lower. In addition, impaired

adaptive behavior, cognitive function and speech and language skills were linked to the

bdnf deficiency (Han et al., 2013; Shinawi et al., 2011). Patients with WAGR syndrome

may have deletions of large numbers of genes, making it difficult to distinguish the effects

of only bdnf deletion. However, symptoms of subjects without WAGR syndrome who

have bdnf deletions support these findings, since the subjects were obese, insensitive to

pain and had neurodevelopmental and behavioral phenotypes (Ernst et al., 2012).

Moreover, in a case study a patient with bdnf haploinsufficiency was obese, hyperactive,

developmentally delayed especially in speech and language skills, and manifested

decreased sensitivity to pain, low IQ, impaired attention and short-term memory

function (Gray et al., 2006).

De novo loss-of-function mutation in TrkB gene was found in an 8-year old boy suffering

from severe obesity and hyperphagia (Yeo et al., 2004). In addition, the boy had

developmental delay especially in speech and language, deficits in short-term memory,

23

stereotypic behavior and impaired nociception. The mutation resulted in replacement of

one tyrosine residue by cysteine in the catalytic domain of the receptor. When studied in

cell culture (PC12 cells) this mutation seemed to decrease the ligand induced

phosphorylation of the receptor and also impaired downstream ERK phosphorylation.

The bdnf haploinsufficient patient basically shows similar phenotypes as the patient with

the loss-of-function mutation in the trkb gene.

An interesting polymorphism in the bdnf gene results from a single amino acid

substitution of valine to methionine in the prodomain of BDNF at codon 66 (BDNF

val66met) (Egan et al., 2003). BDNF met polymorph impairs the episodic memory in

humans. BDNF met polymorphism, however, does not seem to be a risk factor for

depression in humans (Verhagen et al., 2010). In contrast to BDNF haploinsufficiency,

BDNF val66met polymorphism was not associated with obesity or eating disorders

(Friedel et al., 2005). The substitution does not occur in mature BDNF but in the pro-

domain of proBDNF, thus, it does not seem to have a direct effect on the functions of the

mature BDNF, for example on the activation of the TrkB receptor or on the ability of

BDNF to induce neurite outgrowth in PC12 cells (Egan et al., 2003). However, this single

amino acid substitution seems to affect the intracellular processing and activity-

dependent secretion of BDNF.

It has been estimated that 20-30 % of human population is heterozygous for the BDNF

val/met, which means that both Val- and Met-form of BDNF can be expressed. When co-

expressed val- and met-BDNF can form heterodimers affecting the distribution of the

val-BDNF in the cell that results in its increased accumulation in the cell body because

of abnormal sorting from Golgi apparatus to the secretory granules in PC12 cells (Chen

et al., 2004).

In a fear conditioning paradigm human BDNF met allele carriers did not differ from

val/val carriers (Soliman et al., 2010). However, the met allele resulted in impaired fear

extinction that was accompanied by decreased activity in the ventromedial prefrontal

cortex and increased activity of the amygdala in human neuroimaging studies done

during extinction training. In another human imaging study the met allele was associated

with slightly impaired memory retrieval (Hariri et al., 2003). Hippocampal activity was

reduced in subjects with the met allele during memory encoding and retrieval, however,

it has been found that hippocampal volume of the met allele carriers is decreased (Hajek

et al., 2012).

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2.3 NEUROTROPHIN AND NETWORK THEORIES OF ANTIDEPRESSANT ACTION

2.3.1 Antidepressant drugs

The first antidepressant drugs (AD) were serendipitously discovered in the 1950’s. The

monoamine oxidase (MAO) inhibitor iproniazid was tested for treatment of tuberculosis

but was found to improve the mood of depressed patients receiving the medicine

(Castrén, 2005). Iproniazid was soon followed by the first tricyclic antidepressant

imipramine. Because both of these drugs affect the monoamine neurotransmitters it was

suggested that depression may be caused by abnormal levels of monoamines that could

be normalized with antidepressant treatment (Schildkraut, 1965). MAO inhibitors and

tricyclic antidepressants were followed by more selective drugs targeting noradrenergic

or serotonergic systems, for example the selective serotonin reuptake inhibitors (SSRI)

that are widely used today.

In general, antidepressant drugs require weeks of treatment before their therapeutic

effects appear. The primary pharmacological effects of the drugs on monoamines appear

almost immediately suggesting that the therapeutic effects of ADs are not directly

evolving from the increased monoamine levels in the synaptic cleft. Adaptation and

plasticity of the neuronal networks resulting from chronic exposure to ADs could

underlie the slow manifestation of the clinical therapeutic effect of ADs (Castrén, 2013).

It is also interesting that monoamine depletion, which leads to a lack of serotonin or

noradrenaline/dopamine in the brain, cannot produce depressive-like symptoms in

healthy volunteers and does not lower mood in depressed patients (Ruhé et al. 2007).

This suggests that there is no direct correlation with the brain monoamine levels and the

depressed mood. However, the depletion in experimental conditions is acute and it is

possible that longer depletion is required to induce depression symptoms. In subjects

who had been previously treated with antidepressant drugs but are in a remission,

tryptophan depletion moderately decreased the mood whereas phenylalanine/tyrosine

depletion had no significant effect (Ruhé et al., 2007). In patients, who are in remission

and are currently treated with antidepressant drugs, depletion of the corresponding

monoamine in which the drug exerts its main effect can reinstate the depressive

symptoms (e.g. tryptophan depletion in patients using SSRIs) (Ruhé et al., 2007). These

results suggest that depressed patients are more susceptible to depressive symptoms

because of a possible vulnerability in their monoaminergic systems. Interestingly, in

healthy volunteers with a family history of major depressive disorder a slight reduction

in the mood could be achieved with monoamine depletion, suggesting a genetic

component involved in the sensitivity to the effects of monoamine depletion (Ruhé et al.,

2007).

2.3.2 Concept of neurotrophin theory of depression and antidepressant action

Neurotrophin theory of depression postulates that reduction of BDNF expression would

impair the survival and connectivity of neurons resulting in neuronal atrophy, especially

in brain areas vulnerable to stress and involved in the regulation of mood (e.g.

hippocampus) (Duman et al., 1997). The expression levels of BDNF may be promoted

following treatment with antidepressant drugs aiding the neurons in resisting the

negative effects of stress to restore connectivity. In support of the neurotrophin theory

of depression, reduced BDNF levels in the brain and serum of depressed patients, as well

25

as increases in BDNF expression after AD treatment has been reported (Chen et al., 2001;

Karege et al., 2002, 2005; Matrisciano et al., 2009; Shimizu et al., 2003).

In the blood BDNF is found in the platelets and it can be released upon stimulation

induced by stress or injury (Fujimura et al., 2002). Megakaryocytes, which produce the

platelets, express bdnf mRNA in humans and rats but not mice (Chacón-Fernández et

al., 2016). Megakaryocytes also store BDNF protein, which is then transferred to

platelets. Levels of BDNF in serum from depressed patients are found to be lower

compared to healthy controls, with some studies suggesting that BDNF levels are

correlated with the severity of depressive-like symptoms (Karege et al., 2002, 2005;

Pandey et al., 2010). Antidepressants appear to increase BDNF levels in serum since

depressed patients treated with ADs have elevated levels of BDNF in serum compared to

non-treated controls, however, the ability of different ADs to affect BDNF levels seem to

vary (Matrisciano et al., 2009; Shimizu et al., 2003). In support of these findings, in

meta-analyses BDNF concentration has been found to be lower in the serum of depressed

patients than in healthy controls or antidepressant-treated depressive patients, however,

no correlation between BDNF levels and symptom severity was found (Molendijk et al.,

2014; Sen et al., 2008). Low serum BDNF concentration is thought to result from

decreased release of BDNF from the platelets and not from the overall decrease in blood

BDNF levels (Karege et al., 2005).

In addition to blood, changes in brain BDNF levels have been observed in post mortem

studies of depressed patients. Specifically, patients on antidepressant medication have

increased BDNF expression in the hippocampus, whereas suicide victims have reduced

BDNF and TrkB expression in the hippocampus and prefrontal cortex (Chen et al., 2001;

Dwivedi et al., 2003)

2.3.3 Regulation of BDNF and TrkB by stress and antidepressant drugs

The neurotrophin hypothesis of depression is supported by animal studies. Stress and

glucocorticoid injections were first shown to downregulate bdnf mRNA expression in the

hippocampus (Smith et al., 1995a). Stress did not produce changes in cortical bdnf levels

and, in fact increased bdnf expression in the hypothalamus and pituitary gland (Smith et

al., 1995b). The length, timing, and type of stressor affect bdnf expression in a

complicated manner since some bdnf transcripts are downregulated and some

upregulated in specific hippocampal subfields (Nair et al., 2006). Stress-induced

downregulation of bdnf in the hippocampus could be prevented by chronic

antidepressant treatment, electroconvulsive shocks or voluntary exercise (Dwivedi et al.,

2006; Nibuya et al., 1995a; Russo-Neustadt et al., 2001). Chronic AD treatment per se

also increased bdnf and trkb mRNA levels in the hippocampus and the frontal cortex.

The combination of an antidepressant drug and voluntary exercise increased

hippocampal bdnf expression even more than either treatment alone (Russo-Neustadt et

al., 2001). Chronic antidepressant treatments can also prevent the increase in the

immobility time in the forced swim test (FST) and reduction in the sucrose consumption

in the sucrose preference test that can be seen in rodents after chronic stress (Haenisch

et al., 2009).

Important finding linking BDNF and mood came from studies showing that BDNF could

modulate monoaminergic systems that have been strongly related to depression

(Mamounas et al., 1995; Siuciak et al., 1996). It was demonstrated that BDNF infusion

26

for 6-7 days into the midbrain produced antidepressant-like behavior in the learned

helplessness model and the forced swim test in rats; suggesting that BDNF itself was

sufficient to induce antidepressant-like behavioral responses (Siuciak et al., 1997).

Injection of BDNF into the dentate gyrus or CA3 of the hippocampus, or

intracerebroventricular infusion of BDNF also resulted in AD-like behavior in rats

(Hoshaw et al., 2005; Shirayama et al., 2002). Intrahippocampal implant releasing

BDNF during 2 days reduced immobility in the FST when tested 7 days after

implantation (Sirianni et al., 2010). BDNF overexpression in the dorsal dentate gyrus

prevented the reduction in sucrose preference after chronic mild stress paradigm

suggesting that BDNF could increase the resilience to stress (Taliaz et al., 2011).

The antidepressant-like effects of BDNF are likely mediated via its receptor TrkB and it

has been demonstrated that mice overexpressing TrkB show antidepressant-like

behavior in FST (Koponen et al., 2005). TrkB overexpressing mice have increased

baseline activation of the receptor, which could explain the behavioral phenotype.

Supporting this idea, intact BDNF-TrkB signaling is necessary for the behavioral effects

of antidepressant drugs in the FST since BDNF heterozygous knockout mice, BDNF

conditional knockout mice, and mice overexpressing the truncated isoform of the TrkB

receptor do not respond normally to antidepressant drugs (Monteggia et al., 2004;

Saarelainen et al., 2003). Antidepressant drugs with unique pharmacological profiles

activate TrkB receptors and signaling pathways in the mouse brain (Rantamäki et al.,

2007; Saarelainen et al., 2003). Whether ADs activate TrkB through increasing the

release of BDNF or through other mechanisms remains to be investigated.

An often cited paper from Eisch et al. 2003 describes that infusion of BDNF to ventral

tegmental area (VTA) shortens the latency to the first immobility period in FST (Eisch et

al., 2003). However, the number of the rats in the test was really small, only 4-5 animals

per group and there was no effect of BDNF on the immobility time. Therefore the results

generated from this paper do not fully demonstrate the role of BDNF in VTA on

behavioral effects in the FST. It has been later shown that electroconvulsive shock (a

model of electroconvulsive therapy (ECT)) treatment for 10 days increased BDNF

expression in the dorsal dentate gyrus but decreased BDNF expression in the ventral

tegmental area (Taliaz et al., 2013). Knocking down BDNF from the dentate gyrus did

not, however, abolish the antidepressant-like effect of ECT whereas overexpression of

BDNF in the VTA could block the behavioral effects of ECT in the FST. BDNF

overexpression in the VTA reduced sucrose preference and that could be rescued by ECS.

Additionally, BDNF expression in the VTA has been linked to the development of social

aversion in the social defeat stress paradigm. Social defeat stress increased BDNF

expression in the nucleus accumbens and BDNF deletion specifically in VTA (the most

probable source for BDNF in nucleus accumbens) prevented the defeated mice from

developing social aversion behavior (Berton et al., 2006). Chronic social defeat stress

also downregulated bdnf mRNA in the hippocampus affecting especially the

transcription of bdnf variants III and IV (Tsankova et al., 2006). Chronic treatment with

AD imipramine prevented the decrease in bdnf mRNA and also increased bdnf mRNA in

non-stressed mice.

2.3.4 Neurogenesis and depression

The first study showing that chronic (14-28 days), but not single treatment with

antidepressant drugs and electroconvulsive shocks (10 days) increases neurogenesis

27

(proliferation) in the rat brain was done by Malberg et al. (2000). The effect of

antidepressant drugs was specific to the hippocampus since in the subventricular zone

antidepressant drugs had no effect on the neurogenesis. Then, Santarelli et al. (2003)

showed the same effects in mice. X-ray irradiation of the subgranular zone abolished the

behavioral effect of fluoxetine in the novelty suppressed feeding test and also in some

parameters measured after chronic unpredictable stress model indicating the

requirement for neurogenesis in the behavioral effects of fluoxetine. Chronic fluoxetine

treatment increases proliferation, facilitates the maturation and promotes the survival of

newborn cells and it can prevent the stress-induced downregulation of hippocampal

neurogenesis (Alonso et al., 2003; Wang et al., 2008).

Chronic treatment with fluoxetine increases the amount of BrdU positive neurons in the

hippocampus while simultaneously activating apoptosis; suggesting that

antidepressants affect the turnover of newborn cells rather than cell proliferation

(Sairanen et al. 2005). However, after chronic antidepressant treatment the survival of

the new-born cells seems to be increased. Antidepressant-induced proliferation in the

SGZ was unaffected in BDNF heterozygous knockout mice or in mice overexpressing the

truncated TrkB receptor, suggesting that BDNF-TrkB signaling is not critical for

regulating the effects of antidepressant drugs on proliferation of neural precursor cells.

Long-term survival of newborn cells was decreased in BDNF heterozygous knockout

mice or TrkB.T1 overexpressing mice and the antidepressant effect was abolished.

There exists a strong correlation between the time course of the neurogenesis and the

behavioral effects of antidepressant drugs, thus supporting the hypothesis that

neurogenesis could underlie some important aspects of therapeutic effects of

antidepressant drugs (Castrén and Hen, 2013). This is further supported by the findings

from human studies that show an increased expression of neural progenitor cells in the

dentate gyrus of SSRI treated patients compared to non-treated patients or patients

treated with tricyclic antidepressant drugs (Boldrini et al., 2009, 2012). Additionally, the

volume of the dentate gyrus was larger in the SSRI treated patients. In baboon

hippocampus chronic treatment with antidepressant fluoxetine did not affect the

proliferation of adult born neurons (Wu et al., 2014). How ADs exactly promote

neurogenesis is not known. In general the microenvironment around the neural stem

cells (i.e. the neurogenic niche) can regulate the function of the neural progenitor cells,

and it is possible that antidepressants promote expression or availability of some critical

regulators of neurogenesis (Zhao et al. 2008).

Additional plasticity promoting effects are associated with antidepressant drugs. These

include increases in dendritic spines of inhibitory interneurons of PFC and pyramidal

cells of the CA1 and CA3 areas of the hippocampus (Guirado et al., 2014; Hajszan et al.,

2005). Furthermore, antidepressants have been shown to enhance LTP in the medial

perforant path-dentate gyrus pathway and in the lateral amygdala (Karpova et al., 2011;

Wang et al., 2008).

2.3.5 The concept of network theory of depression and antidepressant action The delayed onset of action of antidepressant drugs brought about the idea that the

clinical effects of ADs require adaptation and changes at the neuronal network level

(Castrén, 2013; Castrén and Hen, 2013). The plasticity of the adult brain is restricted,

however, the molecular mechanisms of learning and memory are based on brain

28

plasticity that is expressed as strengthening or weakening of synapses. In addition to

changes in existing synapses, new neuronal connections can be formed when adult born

neurons connect to existing neural networks indicating adult neurogenesis affects

neuronal plasticity (Castrén and Hen, 2013). Plastic changes on the structural or

functional level can eventually result in remodeling of complete neural networks.

Information processing (e.g. emotions) requires the cooperation of multiple brain

regions via neural networks (Castrén, 2005). The information transmitted between brain

areas can be enhanced or diminished as a result of network level plasticity. In depressed

patients abnormalities in morphology and wiring of the areas involved in emotional

processing have been detected (Price and Drevets, 2009). It has been reported that

depressed patients have synaptic atrophy in the dorsolateral PFC and decreased volumes

of PFC and hippocampus (Kang et al., 2012; Savitz and Drevets, 2009). Depression can

be a manifestation of these structural and functional changes in the mood-related neural

networks. Plastic processes involved in the recovery from depression likely affect the

neural networks controlling mood, balancing their actions to resemble the healthy state

(Castrén, 2005).

An important aspect of the network theory of depression and antidepressant action is

that the shaping of the neuronal networks requires environmental guidance (Castrén,

2013). The ability of antidepressant drugs to enhance the plasticity of the brain has been

shown in studies conducted on rat visual cortex and mouse fear processing circuitry

(Karpova et al., 2011; Maya Vetencourt et al., 2008). Results from both studies

demonstrate the requirement for environmental guidance to support antidepressant

effects.

2.3.6 Plasticity models and antidepressant drug action

The antidepressant fluoxetine can restore juvenile-like plasticity in the adult rodent

visual cortex (Maya Vetencourt et al. 2008). This is a crucial finding supporting the

hypothesis that the effects of ADs could be mediated by enhanced plasticity of the

neuronal networks. In the study Maya Vetencourt et al. used two models to examine the

plasticity of the visual cortex: an amblyopia model and monocular deprivation during

adulthood. In the amblyopia model one eye is closed during the critical period of visual

system development, thus resulting in ocular dominance of the open eye in the visual

cortex and reduction in the visual acuity of the closed eye. After the end of the critical

period the connections and acuity of the closed eye cannot be restored anymore.

Similarly, if monocular deprivation is done during adulthood, the connections or acuity

of the closed eye are not weakening since the critical period is over and the state of the

visual cortex and binocularity are already established (Hensch, 2005). However, after

chronic fluoxetine treatment it was possible to modify the connections of the visual

cortex similarly to what could be done during the critical period (Maya Vetencourt et al.,

2008).

In adulthood closing of one eye during fluoxetine treatment resulted in a similar shift in

ocular dominance that can be achieved when the eye is closed during the critical period

(Maya Vetencourt et al., 2008). In the control group, no shift in ocular dominance was

observed. During eye closing the response evoked by the closed eye in the visual cortex

was reduced, demonstrating that the shift in ocular dominance happens because the

connections from the closed eye are weakening and not because the connections from

the open eye would be strengthening. In addition, when the amblyopic rats were treated

29

with fluoxetine the visual acuity of the closed eye improved to the level of the normal eye,

however, it was necessary to close the opposite eye to cause the closed eye to recover.

This is crucial, since it demonstrates the requirement for environmental guidance for the

effects of fluoxetine. Another possibility is a need arising from the environment that

requires adaptation of the connections. If the better eye stays open, the animal can

continue to use it and there is no need to adjust to new situations, but when the better

eye is closed there is a requirement for the weaker eye to try to adapt.

The mechanism underlying how chronic fluoxetine treatment could make the cortex

more plastic was linked to increased expression of BDNF and reduced levels of GABA-

mediated inhibition in the cortex (Maya Vetencourt et al., 2008). These effects are not

dependent on the shift of the ocular dominance, but rather appear as a result of chronic

fluoxetine treatment. As mentioned previously, the fluoxetine-induced shift in ocular

dominance cannot occur without closing of the stronger eye. In the case of monocular

deprivation during adulthood, the shift in ocular dominance occured because of

weakening connections from the closed eye and not because of strengthening of

connections from the open eye (Maya Vetencourt et al., 2008). The functional role of

BDNF as a neurotrophic factor is to promote synaptic strengthening rather that

repression, thus there may be many additional mechanisms contributing to the enhanced

plasticity. BDNF has an important role as a plasticity inducer or enhancer since direct

administration of BDNF via minipumps to the visual cortex at the same time with

monocular deprivation resulted in an ocular dominance shift (Maya Vetencourt et al.,

2008). The likely explanation would be that BDNF via TrkB signaling initiates a process

which renders the synapses in a more plastic state, including the possibility to strengthen

but also to weaken the synapses.

The other mechanism suggested by Maya Vetencourt et al. is reduced GABAergic

inhibition in the cortex. GABAergic neurotransmission is an important regulator of

cortical plasticity and increases in the inhibitory GABAergic signaling are linked to the

closure of the critical period (Hensch, 2005). When diazepam (GABAA receptor positive

allosteric modulator) was infused to the visual cortex during monocular deprivation it

could prevent the shift in ocular dominance caused by fluoxetine (Maya Vetencourt et

al., 2008). This suggests that enhanced GABAergic signaling could block the plasticity

inducing effects of fluoxetine. It would be interesting to know if diazepam could also

block the effects of intracortical BDNF infusion.

Chen et al. studied the effects of fluoxetine on dendritic spine dynamics and found that

fluoxetine treatment, similarly to monocular deprivation alone, increased retraction and

elongation of the spines but not the total spine number in the visual cortex layer 1 and

layer 2/3 interneurons (Chen et al., 2011). In combination with monocular deprivation

fluoxetine increased spine elongations in layer 2/3 during the first two days of monocular

deprivation. This was faster than in the group treated with monocular deprivation alone

without fluoxetine, in which the increase in the spine elongation was seen 4-7 days after

starting the monocular deprivation. The retraction rate, however, was similar in

monocular deprivation groups with or without fluoxetine. The retraction of spines co-

occurred with reductions in the amount of inhibitory synapses onto excitatory cells,

suggesting that monocular deprivation reduces the amount of inhibitory synapses onto

excitatory neurons and could thus decrease the network inhibition. With fluoxetine these

changes happen more quickly indicating that fluoxetine “primes” the visual cortex for

30

monocular deprivation by potentially affecting plasticity enhancing mechanisms and

itself reducing the cortical inhibition (Chen et al., 2011).

The effect of fluoxetine on plasticity was also demonstrated on brain circuits involved in

the fear processing, proving that the effect is not specific for the visual system only. In

Karpova et al. (2011) fear-conditioning was used to study the antidepressant-induced

plasticity of the fear processing circuitry. In fear conditioning paradigm the animal is

exposed to unconditioned noxious stimulus (e.g. electric shock) which is combined with

the conditioned stimulus (e.g. sound) (Maren, 2001). The animal learns quickly that the

conditioned stimulus predisposes the noxious unconditioned stimulus and detecting the

conditioned stimulus leads to freezing behavior. This behavioral response can be

perturbed by exposing the animal to the conditioned stimulus without the unconditioned

stimulus thereby removing the connection between stimuli. However, fear memory

cannot normally be completely removed in adult animals since after a certain period of

time when the animal is again exposed to the conditioned stimulus the fear is renewed

and the animal freezes again after detecting the conditioned stimulus (Myers and Davis,

2006). Interestingly, in young animals fear memory can be permanently removed by

extinction training (Gogolla et al., 2009). This concept was used in the study of Karpova

et al. (2011) to investigate the potential “rejuvenating” effect of fluoxetine in an area

different from visual cortex of adult mice. Fluoxetine indeed facilitated permanent fear

extinction, since the mice treated chronically with fluoxetine showed reduced fear

renewal and fear reinstatement (Karpova et al., 2011). This was repeated in two different

experiments where the mice received chronic fluoxetine treatment either before starting

the fear conditioning paradigm or in a more clinically relevant set up in which the mice

were treated with fluoxetine after fear acquisition. In vehicle treated animals the fear

renewal and reinstatement were clearly detectable.

The effects of fluoxetine were accompanied by a decreased number of parvalbumin

positive cells surrounded by perineuronal nets in the basolateral amygdala and CA1 area

of the hippocampus suggesting that some neurons lost the parvalbumin expression,

which could be considered as a shift toward a more immature state (Karpova et al., 2011).

Also other markers related to the more immature state of neurons were detected in the

amygdala, namely reduction in the expression of potassium-chloride cotransporter 2

(KCC2) and increases in the expression of polysialynated neuronal cell adhesion

molecule (PSA-NCAM). Long-term potentiation was enhanced in the lateral amygdala of

fluoxetine treated animals demonstrating increased synaptic plasticity (Karpova et al.,

2011). As in the study by Maya Vetencourt et al. (2008) BDNF was demonstrated to be

sufficient to induce changes in synaptic plasticity also in this study, since overexpression

of BDNF in amygdala after extinction training prevented fear renewal similarly to

chronic fluoxetine treatment. To further support the BDNF’s role in fear extinction, the

effects of fluoxetine treatment on fear renewal were lacking in BDNF heterozygous

knockout mice (Karpova et al., 2011).

Together these two studies by Maya Vetencourt et al. and Karpova et al. demonstrate that

chronic fluoxetine treatment enhances plasticity in adult rodent brain. In humans the

possible alterations in brain plasticity by antidepressant drugs are harder to evaluate but

some studies have been done. The visual cortex has been used also in humans to study

the effects of chronic antidepressant treatment. Normann et al. (2007) measured visually

evoked potentials (VEPs) in healthy human subjects and in patients with major

31

depression, both receiving chronic treatment with sertraline. They found that the cortical

plasticity induced by prolonged visual stimulus measured as early VEPs was enhanced

in healthy subjects and reduced in depressed patients when compared to healthy

controls. In the study there was no group of depressed patients without antidepressant

treatment, so the effect of antidepressant drugs in depressed patients could not be

demonstrated. According to the results, the ongoing antidepressant treatment could not

increase the VEPs of the depressed patients to normal levels. Some of the patients in the

study were also taking benzodiazepines at the same time with antidepressant drugs, but

the average VEPs were similar in patients with or without benzodiazepines (Normann et

al., 2007). In the rat study by Maya Vetencourt et al. (2008) the benzodiazepine

diazepam could prevent the plastic changes in the visual cortex. Also diazepam blocked

the neurogenesis-inducing effect of chronic fluoxetine treatment (Wu and Castrén,

2009), thus suggesting that simultaneous use of antidepressants and benzodiazepines

may be problematic in depressed patients.

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2.4 RAPID-ACTING ANTIDEPRESSANT DRUGS

2.4.1 Short history of rapid antidepressant effects

Induction of seizure activity in the brain was found to relieve symptoms of psychiatric

patients in the 1930’s. Pharmacologically induced shocks with insulin or pentylentetrazol

were followed and soon replaced by electrically-induced seizures (Merkl et al., 2009).

Electroconvulsive therapy (ECT) was found to be a surprisingly effective treatment for

psychiatric disorders, especially depression. ECT, however, has side effects that include

severe memory problems, thus restricting its use. The emergence of antidepressant drugs

reduced the need for ECT and other shock treatments. ECT, however, is still in use and

is currently the most effective treatment for the patients who do not respond to

conventional antidepressant drugs or psychotherapy (Greenberg and Kellner, 2005;

Group, 2003). The mechanism of action of ECT is not known, but it is thought that

neurotransmitter systems, especially monoamines and GABA are targeted. For example,

ECT is known to increase expression of neurotrophic factors, neurogenesis, and increase

hippocampal volume and connectivity (Abbott et al., 2014; Merkl et al., 2009). Amongst

other things it has been suggested that the post seizure burst suppression is an important

factor determining the efficacy of ECT (Langer et al., 1985). In general, understanding

the mechanism of action of ECT would be beneficial in the development of novel rapid

and effective depression treatments.

2.4.2 The effects of rapid-acting antidepressant ketamine

Ketamine is a non-competitive NMDA receptor antagonist generally used as an

anesthetic. Ketamine acts as an open-channel NMDA receptor blocker inhibiting the ion

flux through the channel (Sanacora and Schatzberg, 2015). NMDA receptors are widely

expressed in the brain and can be found almost in all excitatory synapses (Johnson et al.

2014). NMDA receptor antagonists were found to produce antidepressant-like behavior

in animal models and it was discovered that conventional antidepressant drugs affect the

glutamatergic system with NMDA receptor antagonists having effects that resemble

those of antidepressant drugs (Papp and Moryl, 1994; Skolnick et al., 1996; Trullas and

Skolnick, 1990). These findings further encouraged the testing of ketamine in depressed

humans. The antidepressant effect of a subanesthetic dose of intravenous ketamine

infusion in humans was first characterized by Berman et al. in a small group of depressed

patients; and the findings have been replicated in several studies (Berman et al., 2000;

Murrough et al., 2013; Zarate CA et al., 2006). In human patients the antidepressive

effect of ketamine appears remarkably fast, within a few hours, and the effect of one

administration normally lasts up to one week (McGirr et al., 2015). Ketamine has a short

half-life (about 3 hours) so its long-lasting effects cannot be simply explained by “real-

time” NMDA receptor antagonism.

The finding that noncompetitive NMDA receptor antagonist memantine does not

produce antidepressant effects in human patients suggests that ketamine has some

specific properties that underlie its antidepressive efficacy (Smith et al., 2013; Zarate CA

et al., 2006). The clinical differences in ketamine and memantine could be explained by

their off-target effects (other than NMDA receptors) or by their effects on distinct

populations of NMDA receptors (Johnson et al., 2014). In the absence of Mg2+ both

ketamine and memantine blocked miniature excitatory postsynaptic currents (mEPSCs)

suggesting that both are able to block NMDA receptor-mediated responses, however, in

33

physiological conditions (in the presence of Mg2+ ) only ketamine blocked mEPSC’s

(Gideons et al., 2014). The results obtained on other NMDA receptor antagonists in

animal models also suggest that ketamine has unique mechanisms of action since they

do not seem to produce as fast and long-lasting effects as ketamine (Autry et al., 2011;

Maeng et al., 2008). It has been shown, however, that the NR2B subunit of the NMDA

receptor is crucial in mediating ketamine’s antidepressant-like behavioral effects in

rodents (Miller et al. 2014). Recently, it was shown that a metabolite of ketamine,

(2R,6R)-hydroxynorketamine (HNK) can reproduce the behavioral effects of ketamine

in animal models (Zanos et al., 2016). (2R,6R)-HNK does not bind to NMDA receptors

but increases AMPA-mediated currents. The behavioral effects of (2R,6R)-HNK can be

blocked with pretreatment of 2,3- dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-

7-sulfonamide (NBQX) interestingly even when the NBQX is administered 23,5 h after

(2R,6R)-HNK. This indicates that AMPA receptor-mediated synaptic potentiation could

be maintaining the antidepressive effects of ketamine. Pre-treatment with NBQX can

abolish the antidepressant-like effects of ketamine and another NMDA receptor

antagonist MK-801, but not imipramine, in FST (Autry et al., 2011; Maeng et al., 2008).

The role of AMPA receptors in regulating the effects of ketamine is further supported by

the findings that ketamine increases glutamate levels and can thus promote signaling via

kainate and AMPA receptors. The counterintuitive finding of increased glutamate levels

in the PFC after injection of a subanesthetic dose of ketamine was first described in rats

(Moghaddam et al., 1997). The effects of ketamine on glutamate and GABA

concentrations in the human PFC have also been studied. Increases in GABA and

glutamate/glutamine levels in the anterior cingulate cortex were found in human studies

during ketamine infusion, specifically in the first 30 minutes, and soon after ketamine

infusion (Milak et al., 2015; Rowland et al., 2005; Stone et al., 2012; Taylor et al., 2012).

If GABA and glutamate levels were measured from occipital cortex of depressed patients

3 h or 48 h after ketamine infusion changes were no longer detected (Valentine et al.,

2011). In rats 30 mg/kg ketamine increased GABA, glutamate, and glutamine in the

mPFC 10 minutes after the injection, but not in hippocampus or after ketamine dose of

80 mg/kg (Chowdhury et al., 2012). With glutamate levels increased and NMDA

receptors blocked glutamatergic effects are mediated by kainate and AMPA receptors.

An anesthetic dose of ketamine also increased the extracellular dopamine levels in the

PFC partially in an AMPA/kainate receptor-dependent manner (Moghaddam et al.,

1997).

Riluzole is an NMDA receptor blocker and modulator of glutamate release that has been

shown to increase the expression of AMPA receptors (Dutta et al., 2015). It has been

hypothesized to be able to prolong or enhance the ketamine’s antidepressant effects and

to protect against relapse, especially if the mechanisms of antidepressant effects of

ketamine are increased AMPA-mediated signaling and blockade of NMDA receptors. No

significant change, however, in the duration of antidepressant effects of ketamine has

been observed in humans if ketamine infusion has been followed by treatment with

riluzole (Ibrahim et al., 2012; Mathew et al., 2010).

Increases in BDNF protein expression and TrkB signaling has been also linked to the

antidepressant effects of ketamine in rodents (Autry et al., 2011). Autry et al. (2011)

found in their study that ketamine (3mg/kg) rapidly increases protein translation via

dephosphorylation of eukaryotic elongation factor 2 (eEF2). They show that the

34

translation of mBDNF and proBDNF is significantly increased 30 min after

administration of ketamine or another NMDA-receptor antagonist MK-801.

Furthermore, they demonstrate that blockers of the eEF2 kinase, which inhibit the

phosphorylation of eEF2 by the kinase, have similar effect as ketamine in FST and with

regards to BDNF protein levels. The AD effects of ketamine and eEF2 kinase inhibitor

were abolished in the inducible BDNF conditional knockout mice in FST, and for

ketamine also in TrkB conditional knockout mice indicating that BDNF-TrkB signaling

is involved in the behavioral effects of ketamine (Autry et al., 2011). However, in BDNF

heterozygous knockout mice ketamine produced an antidepressant-like effect in the FST

similarly to wild type mice (Lindholm et al., 2012). In rats, acute administration of

ketamine (15 mg/kg) increased BDNF protein levels in the hippocampus but chronic

administration of ketamine with the same dose for 14 days did not (Garcia et al., 2008a,

2008b). Chronic administration of ketamine with 5, 10, or 15 mg/kg doses, however,

decreased immobility time in FST.

The effects of ketamine in mice and humans with the BDNF val66met polymorphism

indicate that the activity-dependent release of BDNF could be important in regulating

the effects of ketamine. BDNF met/met mice have decreased spine density and spine

head diameter in the layer V pyramidal cells of PFC and the ketamine-induced increase

in spine number is abolished in these mice (Liu et al., 2012). BDNF met/met mice did

not respond to ketamine when tested 24h post-injection (10 mg/kg) in FST. In a study

by Laje et al. (2012) depressed human patients carrying the met allele (met/met or

val/met) did not respond to ketamine as effectively as the val/val carriers (24 %

reduction in the Hamilton depression rating scale scores in met group and 41 % for

val/val) when measured 210-230 minutes after ketamine infusion (Laje et al., 2012).

Infusion of BDNF function blocking antibody to the mPFC of rats 30 minutes before

ketamine administration (10mg/kg) abolished the behavioral effects of ketamine in the

FST 24 hours after the ketamine treatment (Lepack et al., 2014). Also pretreatment with

L-type calcium channel blockers nifedipine or verapamil abrogated the effect of ketamine

in the FST.

In addition to rodent studies, the effects of ketamine on blood BDNF levels have been

assessed in humans. After single ketamine infusion the responding patients showed

higher increase in blood BDNF levels and the BDNF increase correlated with slow wave

activity in electroencephalography during sleep (Duncan et al., 2013). The authors

hypothesized that the increased slow wave activity in ketamine responders is a

consequence of BDNF-induced synaptic strengthening and enhanced plasticity. A

significant negative correlation between Montgomery-Åsberg depression rating scale

score and plasma BDNF concentration in ketamine treated patients, but not in patients

treated with benzodiazepine midazolam, was found when studied 240 min after

ketamine infusion (Haile et al., 2014). In some studies, however, the BDNF levels were

not affected by the ketamine treatment (Machado-Vieira et al., 2009).

It has been demonstrated that ketamine activates the Akt/ERK-mTOR-p70S6kinase

pathway rapidly and transiently and increases the levels of synaptic proteins in 2 h after

the administration (Li et al., 2010). Ketamine also rapidly increased (in 24 h) dendritic

spines in PFC layer V pyramidal neurons and produced antidepressant-like effects 24 h

after administration in FST, novelty suppressed feeding and learned helplessness

paradigms. The effects on synaptic proteins, dendritic spines and behavior could be

35

blocked by intracerebroventricular injection of mTOR inhibitor rapamycin, suggesting

that mTOR signaling is involved in these actions of ketamine.

In addition to mTOR signaling, glycogen synthase kinase 3 beta (GSK3β)-related

mechanisms have been implicated in the action of ketamine. Previously, dysregulation

of GSK3β has been linked to the pathophysiology of depression and the mechanism of

action of mood stabilizer lithium (Jope and Roh, 2006). Ketamine (10mg/kg) increases

GSK3β phosphorylation, which results into inhibition of its function, in the hippocampus

and cortex (Beurel et al., 2011). Interestingly, memantine also has similar effects on

GSK3β as seen with ketamine. Using transgenic mice with mutations in the inhibitory

GSK3α/β phosphorylation sites Beurel et al. (2011) were able to demonstrate that in

these mice the antidepressant-like effects of ketamine in the learned helplessness model

were abolished.

The ability of ketamine to affect fast synaptic transmission and activity of neuronal

circuits could underlie its rapid antidepressant effects. Recently, ketamine-like effects on

rat behavior in FST and NSF were achieved by optogenetic stimulation of infralimbic

cortex (Fuchikami et al., 2015). Interestingly, the antidepressant-like effects achieved

with the optogenetic stimulation of mPFC remained for almost three weeks in FST. The

optogenetic stimulation also caused increase in the amount of dendritic spines in the

infralimbic cortex. In addition the effects of ketamine were blocked by infusing the GABA

agonist muscimol to the infralimbic cortex and direct injection of ketamine to infralimbic

cortex could produce antidepressant-like behavior in the FST and NSF, however, only at

certain dose (10 ng) (Fuchikami et al., 2015). These findings support the hypothesis that

increased activity in a certain population of neurons in PFC resulting from ketamine

induced disinhibition could underlie the antidepressant-like effects of ketamine. It is

possible that in the case of low-dose ketamine treatment specifically the fast-spiking and

tonically active GABAergic interneurons are sensitive to the NMDA receptor blockade by

ketamine, which then via disinhibition produces glutamate release and neuronal

excitability in pyramidal neurons. In pyramidal neurons AMPA and kainate receptors

may mediate the effects of glutamate. Administration of MK-801 (0,1mg/kg) to rats

reduced the firing rate of the fast-spiking interneurons and increased the firing rate of

the regular spiking neurons, suggesting that the cortical excitation caused by NMDA

antagonists results from disinhibition (Homayoun and Moghaddam, 2007). It is

important to note, however, that blocking the NMDA receptors results in complex

network effects that differ also along the timescale of the blockade.

In another recent paper projections from ventral hippocampus to PFC seemed to be

important for the sustained (1 week) effects of ketamine in the FST (Carreno et al. 2015).

K252a infusion to ventral hippocampus blunted the effects of ketamine at the 1 week

timepoint (30 min timepoint results were not studied or shown in the article). Infusion

of K252a to hippocampus blocks TrkB receptor activation in hippocampus, but how that

affects the activity of the cells in PFC was not studied (Carreno et al., 2015). This

demonstrates that Trk receptor activation in the hippocampus by ketamine is important

for sustained antidepressant effects. Optogenetic inactivation of ventral hippocampus

during the ketamine administration did not affect swimming behavior 1 week later but

increased immobility if done during the FST, indicating that ventral hippocampus-PFC

circuitry seems to be involved in antidepressant-like behavioral effects of ketamine

(Carreno et al., 2015). There is also some support for ketamine’s effects on hippocampus-

36

PFC circuit from human studies since immediately after ketamine infusion the functional

connectivity between hippocampus and dorsolateral PFC is increased in healthy human

subjects (Grimm et al., 2015).

The ketamine infusion can produce hallucinogenic effects and acutely impair verbal

learning (Murrough et al., 2014). Abusers who have frequently (about 5 times a week)

used ketamine for long time have decreased hippocampal function when tested on spatial

navigation tasks, spatial memory tasks and pattern recognition memory tasks (Morgan

et al., 2010, 2014). Chronic ketamine use can also increase the risk for cystitis and other

related problems in the urinary tract (Middela and Pearce, 2011; Muetzelfeldt et al.,

2008). The doses used by recreational ketamine users are, however, different than the

dose of ketamine used in the treatment of depression. The most common side effects

after ketamine infusion include nausea, visual disturbances and dizziness (Francois et

al., 2015).

2.4.3 The effects of other rapid-acting antidepressant drugs

The concerns about the therapeutic use of ketamine in the treatment of depression

emerge from the current lack of knowledge about the proper treatment protocol, long-

term effects and dependence and abuse potential of ketamine (Sisti et al., 2014). Thus,

the potential of other NMDA receptor antagonists and anesthetics in the treatment of

depression are of interest. Sub-anesthetic doses of nitrous oxide (laughing gas), which

also acts as NMDA receptor antagonist, has promoted antidepressant-like effects in a

small pilot study done on treatment resistant depressed patients (Nagele et al., 2015).

Also NMDA receptor glycine binding site partial agonist GLYX-13 rapidly alleviated

depression symptoms in depressed patients (Preskorn et al., 2015)

Before ketamine was tested for treatment of depression, studies about antidepressant

potential of isoflurane anesthesia were conducted (Langer et al., 1985, 1995). The

rationale for the experiments came from finding that under isoflurane anesthesia

patients demonstrate similar burst suppression in the electroencephalogram as recorded

during ECT (Langer et al., 1985). The efficacy of isoflurane in treatment resistant

depressed patients was found to be similar to ECT in these small clinical trials and the

finding was recently replicated (Langer et al., 1985, 1995; Weeks et al., 2013). Isoflurane

treatment, however, did not produce as severe cognitive side effects as seen with ECT.

The potential of isoflurane in the treatment of depression has not been widely studied

and there are additional studies with controversial findings about the efficacy of burst

suppression anesthesia in the treatment of depression (García-Toro et al., 2001). It

remains to be studied if burst suppression is the critical factor in the antidepressant

effects of anesthesia or ECT and if anesthesia could be used in the treatment of

depression.

In addition to anesthetics, the cholinergic muscarinic receptor antagonist scopolamine

and serotonin agonist psilocybin have demonstrated rapid antidepressant effects in

depressed patients (Carhart-Harris et al.; Drevets and Furey, 2010; Furey and Drevets,

2006). The pharmacological effects of these drugs differ from those of ketamine,

isoflurane and nitrous oxide indicating that the mechanisms underlying rapid

antidepressant effects are complex and require further characterization.

37

3. AIMS OF THE STUDY

Pharmacological enhancement of brain plasticity could be beneficial in the treatment of

psychiatric disorders, especially depression. Since TrkB receptor is involved in regulation

of brain plasticity it could be a favorable target for drug development. Our purpose was

to develop a method that would be suitable for screening of novel TrkB receptor

activators or inhibitors or molecules that would enhance the effects of BDNF. In addition,

we aimed at characterizing further the mechanisms how the already existing TrkB

activators, the monoaminergic antidepressant drugs, promote TrkB activation. Recently,

TrkB receptor has been also linked to the rapid antidepressant-like effects of ketamine.

In addition to ketamine, isoflurane anesthesia has been demonstrated to rapidly reduce

depression symptoms of treatment resistant depressed patients. Our aim was to examine

molecular, structural and behavioral effects of isoflurane in rodents to elucidate the

putative neurobiological basis of the antidepressant effects of isoflurane.

The specific aims of the study were:

1. To develop novel methods to screen for drugs regulating TrkB receptor

phosphorylation (I)

2. To elucidate the mechanisms of antidepressant-induced TrkB activation

(II,III)

3. To investigate the neurobiological basis of antidepressant effects of isoflurane

anesthesia (IV)

38

4. MATERIALS AND METHODS

The materials and methods personally used by the author are summarized here. Detailed

information of the additional materials and methods can be found in the original

publications.

4.1 Animals

Adult (2-4 months) or pup (P5-P21) C57BL/6JRccHsd mice (Harlan Laboratories,

Venray, Netherland), Bdnf+/−, Bdnf−/−, B6.Cg-Tg(Thy1- YFPH)2Jrs/J (Thy1-YFP,

Jackson Laboratories, Bar Harbor, ME, USA) and transgenic mice overexpressing flag-

tagged TrkB or truncated TrkB.T1 and their wild type littermates were maintained in the

animal facility of University of Helsinki, Finland, under standard laboratory conditions

(21°C, 12-hour light-dark cycle, lights on at 6 A.M.) with free access to food and water.

Littermates were randomly assigned to different treatment groups. All the experiments

were carried out according to the guidelines of the Society for Neuroscience and were

approved by the County Administrative Board of Southern Finland.

4.2 Drug treatments

Fluoxetine (HCl salt; Orion Pharma, dissolved in 0,9% NaCl, 30 mg/kg) was

intraperitoneally injected to mice and the mice were culled 1 h later (II, SDS-PAGE

zymography). Isoflurane (Vetflurane®, Virbac) anesthesia was induced with 4%

isoflurane for 2 min, after which the mouse freely inhaled isoflurane (3,0 % for 1 min,

then 2 % for maximum of 30 minutes; airflow: 0.3-0.5 l/min) in the chamber of mask.

Halothane anesthesia was conducted with similar concentrations and protocol as

isoflurane anesthesia. Body temperature was maintained with a heat pad throughout the

treatment. Sham mice were kept in the induction chamber for 2 min without isoflurane.

NBQX (2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide, Tocris

Bioscience, Bristol, UK) was injected (i.p., 10 mg/kg; dissolved in saline) 10 min before

sham/isoflurane treatment. Mice were culled by cervical dislocation while still under

anesthesia or following the described recovery periods after stunning with CO2. Control

mice were culled by cervical dislocation after stunning with CO2.

4.3 Cell culture

4.3.1 Fibroblasts

TrkB expressing MG87 fibroblasts were cultured in Dulbecco's Modified Eagle Medium

(DMEM) supplemented with 10% fetal calf serum (FCS), 1% penicillin/streptomycin, 1%

l-glutamine and 400 μg/ml G418. The cell line was maintained in a cell incubator (5%

CO2, 37 °C).

4.3.2 Primary neuronal cultures

For the primary neuronal cultures, hippocampi or cortex was dissected from E18 rat

embryos and the tissue dissociated in papain solution (in mg: 10 DL-Cysteine-HCl, 10

BSA, 250 glucose, ad 50 ml PBS (pH 7.4); 10 min, 37 °C). Next the cells were triturated

and suspended in a medium containing 9.8 ml of Ca2+/Mg2+ free HBBS, 1 mM sodium

39

pyruvate, 10 mM HEPES (pH 7.2) and 10 μl DNAse I. The cells were plated onto poly-L-

lysine (Sigma–Aldrich) coated cell culture plates. Neurons were maintained in

Neurobasal medium supplemented with 2% B27, 1% penicillin/streptomycin and 1% L-

glutamine and fresh medium was added twice a week.

4.4 Enzyme-linked immunosorbent assay (ELISA)

4.4.1 Conventional pTrkB ELISA

Cell homogenates were transferred from culture plates into pre-coated (sc-11-R, Santa

Cruz Biotechnology, Santa Cruz, CA, USA; 1:500–1000 in Optacoat™; overnight at +4

°C) and pre-blocked (2% bovine serum albumin (BSA)/phosphate buffered saline-Tween

20 (PBS-T); 2 h at room temperature) white 96-well Optiplate™ (PerkinElmer Oy,

Espoo, Finland) plates and 3% BSA/PBS-T (+2 mM Na3VO4 tyrosine phosphatase

inhibitor) added ad 200 μl. After overnight incubation at +4 °C the wells were washed

with PBS-T (4 × 300 μl) and anti-phosphotyrosine antibody was added to the wells (in

house biotinylated PY20; AbD Serotec, Kidlington, UK; 1:1000 in 2% BSA/PBS-T;

overnight at +4 °C). Following sequential washes and horseradish peroxidase (HRP)-

coupled streptavidin antibody incubation (1:10,000 in 2% BSA/PBS-T; O/N at 4 °C) 100

μl of enhanced chemiluminescence (ECL) substrate mix was added to the wells and

luminescence measured with Varioskan Flash plate reader (Thermo Fisher Scientific

Oy).

4.4.2 In situ TrkB ELISA

For the in situ phospho-Trk ELISA, dissociated cells were directly plated onto UV-

sterilized ELISA plates pre-coated (sterile filtered sc-11-R, Santa Cruz Biotechnology,

Santa Cruz, CA, USA; 1:500–1000 in Optacoat™; overnight at +4 °C) and pre-blocked

(sterile filtered 2% BSA/PBS-T; 2 h at room temperature). After drug and/or BDNF

treatment, the plates were put on ice, medium discarded and lysis buffer applied (10–25

μl). Next, the plates were rigorously shaken in a cold room for 30–60 min (800 rpm,

Labsystems Wellmix, Thermo Fisher Scientific Oy) after which 3% BSA/PBS-T (+2 mM

Na3VO4 tyrosine phosphatase inhibitor) was applied ad 200 μl (96-multiwell) or ad 50

μl (384-multiwell). After overnight incubation at +4 °C, the wells were washed and

ELISA assay continued as described above.

4.5 Proof-of-concept small molecule screening

For the small molecule screening, MG87-trkB fibroblasts were cultured directly on 96-

well ELISA plates. Next day, the cells (at ~80 % confluency) were pre-stimulated with

vehicle or with Spectrum Collection compounds for 15 min (5 μM; n = 1; +37 °C) using

Biomek FXp workstation (Beckman Coulter Finland Oy, Helsinki, Finland) and then

post-stimulated with vehicle or with BDNF (5 ng/ml; +37 °C) for another 15 min using

Multidrop Combi (Thermo Fisher Scientific Oy). After the stimulations, the medium was

discarded, cells were lysed in ProteoJET™ membrane protein extraction buffer and

ELISA analyses run as described. Compounds regarded as hits (response to BDNF ± 3×

standard deviation (STDEV)) were re-tested in triplicates and the compounds that still

passed the hit criteria were analyzed in a dose-response assay (0.25, 1, 5, 25 μM; n = 3)

(inhibitors with and activators without BDNF post-treatment).

40

4.6 Brain sample collection

The brain samples were dissected on a cooled dish and homogenized in NP buffer

(137mM NaCl, 20mM Tris, 1% NP-40, 10% glycerol, 48mM NaF, H2O, Complete

inhibitor mix (Roche), 2mM Na3VO4). After minimum of 15 min incubation on ice,

samples were centrifuged (16000 x g, 15 min, +4 °C) and the resulting supernatant was

collected for further analysis. Sample protein concentrations were measured using Bio-

Rad DC protein assay (Bio-Rad Laboratories, Hercules, CA).

4.7 Ex vivo stimulations

The ex vivo BDNF stimulation assay was performed according to Knüsel et al (1994) with

slight modifications. After dissection the samples were chopped to microslices with

scalpel. The slices were transferred in fresh tubes with 10% FCS in supplemented

neurobasal medium and centrifuged (6000 x g, 1 min, +4 °C). Fresh medium was added

to the slices according to their weight and the slices were resuspended to the medium

and divided to stimulation tubes. The medium was removed and 300 µl of prewarmed

(+ 37 °C) neurobasal medium +10% FCS with or without BDNF (Peprotech) or NGF

(Promega) was added. The tubes were closed and incubated at +37°C for 15 minutes

gently shaking. Finally the tubes were put on ice, spun down, the medium removed, the

pellet was rinsed once with PBS and then the samples were homogenized in NP++ buffer.

4.8 Western blot

Western blotting analysis was conducted from medial prefrontal cortex (including

prelimbic and infralimbic cortices), somatosensory cortex and the whole hippocampus.

Immunoprecipitation (500 μg protein) was carried out using anti-Flag M2 antibody

(F1804; Sigma-Aldrich). For immunoprecipitation the samples (500 µg protein) were

rotated overnight with 5 µl of the antibody at +4°C, then 30 µl 50% G-sepharose slurry

was added (rotation 2 h at +4°C) and the precipitated samples were washed 3x500µl PBS

+2 mM Na3VO4). The samples were heated in Laemmli buffer for 5 min at +100 °C.

Immunoprecipitated or unprocessed samples (50 μg protein) were separated with SDS-

PAGE under reducing conditions and blotted onto a PVDF (polyvinylidene difluoride)

membrane (300mA, 1 hour, 4°C). Membranes were incubated with the following primary

antibodies: anti-p-TrkBY816 (1:1000; from Dr. Moses Chao, Skirball Institute, NY, USA),

anti-p-TrkBY816 (#4168; 1:1000; Cell signaling technology (CST)), anti-p-TrkA/BY490/Y515

(#9141; 1:1000;CST), anti-p-TrkA/BY674-5/Y706-7 (#4621S; 1:1000; CST), anti-TrkB

(1:2000; BD Transduction Laboratories, San Jose, CA, USA), anti-p-CREBS133 (#9191S;

1:1000; CST), anti-p-AktThr308 (#4056S; 1:1000; CST), anti-p-mTORS2481 (#2974S;

1:1000; CST), anti-p-p70S6KT421/S424 (#9204S; 1:1000; CST), anti-p-4E-BP1T37/46 (#2855;

1:1000; CST), anti-p-GSK3βS9 (#9336; 1:1000; CST), anti-p-eEF2T56 (#2331; 1:1000;

CST), anti-Trk (sc-11; 1:1000; Santa Cruz Biotechnology (SCB)) and anti-GAPDH (sc-

25778; 1:10000; SCB). Further, the membranes were washed with TBS/0.1% Tween

(TBST) and incubated with horseradish peroxidase conjugated secondary antibodies

(1:10000 in non-fat dry milk, 1 hour at room temperature; Bio-Rad). After subsequent

washes, secondary antibodies were visualized using enhanced chemiluminescence (ECL

Plus, ThermoScientific, Vantaa, Finland) and detected by Fuji LAS-3000 camera (Tamro

Medlabs, Vantaa, Finland).

41

4.9 SDS-PAGE zymography

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) -zymographty

was used to examine the activity of tissue plasminogen activator (tPA). For tPA activity

assay, freshly dissected brain samples were homogenized into buffer consisting of 0.1 M

Tris-HCl (pH 8.0), 2.5% Triton-X-100, 10 µM leupeptin, 10 µg/ml aprotinin, 1 mM

phenylmethanesulfonylfluoride (PMSF). Samples and controls (human recombinant

tPA) were loaded under non-reducing conditions in SDS-PAGE gels ±human

plasminogen (Sigma-Aldrich) and pre-heated non-fat dry milk at run at low current

(∼15–20 mA) overnight (O/N) at cold bath. Next the gels were rinsed thoroughly with

2.5% Triton X-100 to remove SDS and allow proteins to renaturate. Then the gels were

rinsed thoroughly with 10 mM CaCl2 in 50 mM Tris-HCl (pH 7.6) to remove Triton X-

100 and the caseinolysis was allowed to occur by incubating the gels at +37°C for 16–24

h in the same solution. Caseinolytic areas were shown as translucent areas when the gels

were stained with Coomassie Brilliant Blue.

4.10 Immunohistochemistry and dendritic spine analysis

The density of dendritic spines was analyzed in fixed cortical sections from B6.Cg-

Tg(Thy1- YFPH)2Jrs/J (Thy1-YFP, Jackson Laboratories, Bar Harbor, ME, USA) mice.

Animals were transcardially perfused at 24 hours after isoflurane/sham treatment under

deep pentobarbital anesthesia with 4% paraformaldehyde in phosphate buffer (PB) 0.1M

and post-fixed for 1 hour. Floating sections (50 μm thick) were cut using a vibratome (VT

1000S, Leica, Germany) and processed for immunohistochemistry as follows: PBS wash,

blocking for 1 h (10% normal goat serum in PBS/0,2% Triton-X100), anti-YFP antibody

overnight (chicken polyclonal 1:1000; Abcam), PBS wash, Alexa-488-conjugated goat

secondary antibody for 2 h (1:200; Invitrogen), PB wash and mounting using

fluorescence medium (Dako). Images were obtained using a confocal microscope (Leica

TCS SP5II HCS), and pyramidal neurons from the medial prefrontal cortex (mPFC) and

somatosensory cortex (SSCx) were selected with the following criteria: intense

fluorescence, soma located in layer V, and primary apical dendrite >200 μm long. We

imaged the dendrites in three ~65 μm long segments (proximal/medial/distal). We

distinguished different types of dendritic spines: (i) stubby (protrusion length <1.5 μm);

(ii) mushroom (clearly visible head with a diameter >1.5 times the average length of the

neck, and the total length of the protrusion <3 μm) and; (iii) filopodia/thin (the length

of the protrusion >3 μm or non-headed 1.5-3 μm protrusion).

4.11 Quantitative real-time polymerase chain reaction (qPCR)

RNA was extracted using Trizol® reagent (Thermo Scientific) and treated with DNAse I

mix (Fermentas GmbH) and then reverse transcribed using oligo(dT) primer and

RevertAid First Strand cDNA synthesis kit (Thermo Scientific). The control reactions

without reverse transcriptase were also performed. The amount of cDNA was quantified

using Maxima SYBR green qPCR master mix (Thermo Scientific) by real-time PCR. Total

Bdnf cDNA was amplified using the following primers: 5´-

GAAGGCTGCAGGGGCATAGACAAA-3´ and 5´-TACACAGGAAGTGTCTATCCTTATG-

3´. For normalization GAPDH cDNA levels were analyzed with the following primers 5 ́-

GGTGAAGGTCGGTGTGAACGG-3´ and 5 ́-CATGTAGTTGAGGTCAATGAAGGG-3´. Ct

42

values from each sample were obtained using the LightCycler 480 software (Roche

Diagnostics Ltd.).

4.12 Behavioral experiments

4.12.1 Forced swim test

For the forced swim test a mouse was placed into a glass cylinder (diameter 19 cm, height

24 cm) filled with water (21±1°C) to the height of 14 cm. The latency to the first

immobility period (passive floating, when the animal was motionless or doing only slight

movements with tail or one hind limb) and immobility during the last 4 minutes of the 6

min test was measured.

4.12.2 Open field test

The open field test was performed for 30 min in an illuminated (300 lux) transparent

acrylic cage (length 28.5 × height 8.5 × width 20 cm) (Med Associates). Interruptions of

infrared photo beams were used to calculate the overall distance traveled (cm) and time

spent in the center of the arena.

4.12.3 Water maze

Noldus EthoVision XT 10 program (Noldus Information Technology, The Netherlands)

was used for monitoring the mouse behavior in the water maze test. The system consisted

of a black circular swimming pool (Ø 120 cm) and a transparent escape platform (Ø 10

cm) submerged 0.5 cm under the water surface in the centre of one of four imaginary

quadrants. The animals were released to swim (in random positions) from all three other

quadrants facing the wall and the time to reach the escape platform (maximum time 60

s) and the swimming distance were measured in every trial. Two training blocks

consisting of three trials each were conducted daily. The interval between trials was 4-5

min and between training blocks about 4 hours. The hidden platform remained in a

constant location for 3 days (6 initial training sessions) and was thereafter moved to the

opposite quadrant for 2 days (4 reverse training sessions).

4.13 Statistical tests

The statistical analyses were conducted with unpaired two-tailed Student’s t-test, Mann

Whitney U test (non-normally distributed data), one-way analysis of variance (ANOVA)

or two-way ANOVA. Tukey HSD, Newmann-Keuls or Dunnett’s test were used for post

hoc analysis. Statistically significant p value was set to ≤ 0.05. The results are

represented as mean ± SEM (standard error of mean).

43

5.RESULTS

5.1 Development of phospho-Trk ELISAs (I)

Enzyme-linked immunosorbent assay (ELISA) is a widely used method in molecular

biology. Sandwich ELISA is a method conducted on a multiwell plate where capture

antibody is immobilized to the bottom of the plate. After sample incubation the non-

specifically bound proteins are washed away and a secondary antibody coupled to an

enzyme is added. The amount of the protein of interest captured by the primary antibody

can then be measured by adding a substrate to the enzyme and detecting the enzymatic

reaction by colorimetric or luminometric assays. We set up a conventional sandwich

ELISA where we use Trk receptor antibody as a capturing antibody and biotinylated

phosphotyrosine antibody as a secondary antibody. A HRP-coupled streptavidin

antibody is used as a tertiary antibody and after adding substrate to the HRP the amount

of tyrosine phosphorylated Trk receptors can be measured. This conventional phospho-

Trk ELISA was used in publications I,II and III in several experimental conditions to

detect the phosphorylated Trk receptors from cultured neurons and MG87-TrkB cells.

To optimize the ELISA protocol we tested different coating conditions, primary

antibodies and lysis buffers and found that Optacoat buffer, SC-11 rabbit polyclonal Trk

antibody and ProteoJet lysis buffer significantly increased the signal to noise ratio when

compared to other buffers or antibodies (I).

Next, we modified the standard sandwich pTrk ELISA to an in situ format where all the

steps including cell culture, stimulation, antibody incubation and detection can be done

in the same plate (I). Transferring cell lysate from the cell culture plate to the ELISA plate

is time consuming and laborious and can be avoided in the in situ ELISA. In the in situ

pTrk ELISA MG87 fibroblasts overexpressing TrkB receptors are cultured on UV-

sterilized OptiplateTM HB 96 or 384 well plates that are previously coated with SC-11 Trk

antibody and blocked. The cells are incubated 24 hours after which they can be

stimulated. After the stimulation lysis buffer is added to the wells and the plate is

vigorously shaken at + 4 OC for 1 h. When the cells have been lysed the ELISA protocol is

continued similarly to the conventional pTrk ELISA.

We also showed that the in situ ELISA can be upscaled from 96 to 384 well plate making

it suitable for high throughput screening (I). As a proof of concept we performed a

screening of a library of 2000 compounds and identified several compounds that were

able to induce TrkB phosphorylation or inhibit or potentiate BDNF-induced TrkB

activation. Among the TrkB activators was betamethasone valerate and epoxygedunin

and among the inhibitors for example tomatine, gramicidin and fenbendazole.

44

5.2 Mechanisms of antidepressant induced TrkB activation - BDNF and

serotonin transporter (SERT) are dispensable for TrkB activation by

antidepressant drugs (II)

Saarelainen et al. (2003) first described that antidepressant drug imipramine can induce

TrkB receptor phosphorylation in the mouse hippocampus and frontal cortex and later

it was demonstrated that TrkB activation appears to be a common feature of ADs with

different pharmacological properties (Rantamäki et al., 2007). How ADs promote TrkB

activation was, however, unknown and especially it remained unclear whether BDNF is

required for the effect. It is impossible to directly measure BDNF release in the brain and

thus TrkB phosphorylation has been used as a surrogate measure for BDNF release. TrkB

activation can occur, however, independently of BDNF via transactivation (Lee and

Chao, 2001). It has been also suggested that antidepressant amitriptyline and 7,8—

dihydroxyflavone are direct TrkB receptor agonists (Jang et al., 2009, 2010).

In addition to the full-length TrkB receptor (145 kDa), ADs seem to induce

phosphorylation of a protein ~105 kDa in size (II). It has been suggested to be an

immaturely glycosylated form of the TrkB receptor and indeed we found that digestion

with endoglycosidase H, which deglycosylates proteins, reduced the molecular weight of

the 105 kDa protein (II). To support the assumption that the 105 kDa protein is an

isoform of TrkB, in TrkB overexpressing mice also the baseline phosphorylation of this

protein is increased. Furthermore, 1NaPP1 inhibitor treatment of TrkBF616A mutant mice

decreased also the imipramine-induced phosphorylation of the 105 kDa protein.

The TrkB activation occurs in 30 minutes after imipramine injection but no changes have

been detected in the BDNF protein or mRNA levels after acute AD treatment, suggesting

that local release of BDNF could mediate the effect (Nibuya et al., 1995b; Rantamäki et

al., 2007; Saarelainen et al., 2003). It has been shown in hippocampal slices that

proBDNF and BDNF can be released together with tPA in response to increased neuronal

activity (Pang et al., 2004). tPA cleaves proBDNF to BDNF and thus could increase the

BDNF concentration in the synaptic cleft resulting in increased TrkB phosphorylation.

We studied with SDS-PAGE zymography if fluoxetine treatment (1h) increases the

enzymatic activity of tPA in the hippocampus but no difference in the tPA activity or

BDNF protein levels were found between saline and fluoxetine treated animals (II). More

importantly, imipramine activated TrkB in the brains of BDNF cKO animals whose

hippocampal BDNF levels are below detection limits. This indicates that antidepressants

can activate TrkB receptor also in the absence of BDNF.

Since the TrkB receptor activation by ADs occurred independently of BDNF we

investigated if ADs would facilitate adenosine receptor –mediated TrkB transactivation

(II). Previously, adenosine A2A receptors have been shown to transactivate TrkB

receptors in vitro and in vivo (Lee and Chao, 2001; Wiese et al., 2007). Blocking

adenosine A2A receptors, however, did not affect the ability of imipramine to induce TrkB

phosphorylation suggesting that adenosine mediated transactivation is not involved in

the antidepressant-induced TrkB activation (II). To study the possibility that ADs act as

direct agonists of TrkB receptor and can induce the TrkB phosphorylation similarly to

BDNF, we stimulated hippocampal and cortical neuron cultures with imipramine or

45

amitriptyline but no increase in TrkB phosphorylation was detected (II). These results

indicate that antidepressants apparently are not able to directly bind to TrkB receptors.

SERT is the main pharmacological target for fluoxetine and we examined if the effects of

fluoxetine on TrkB phosphorylation require binding to SERT. In the SERT knockout

mice fluoxetine induced TrkB phosphorylation similarly to wild type mice, indicating

that SERT is not required for fluoxetine to activate TrkB receptors (II). Moreover, the

extracellular 5-HT concentration in SERT knockout mice is significantly increased but

no change at baseline TrkB phosphorylation was detected, suggesting that 5-HT does not

directly induce TrkB activation. We further examined the effects of monoamines on TrkB

phosphorylation in cortical neuron cultures but 15 minutes incubations with 10 nM-10

µM concentrations of noradrenaline or 5-HT did not affect TrkB phosphorylation.

5.3 Developmental regulation of TrkB activation by antidepressant drugs and

BDNF (III)

Previously it has been shown that BDNF-induced TrkB phosphorylation is

developmentally regulated (Knusel et al., 1994). We were able to reproduce these

findings using BDNF ex vivo stimulation in microslices prepared from mouse

hippocampus of different developmental stages (P5-P21) demonstrating that

responsiveness of TrkB to exogenous BDNF decreases dramatically after P12 (III).

Intriguingly, we further discovered that at this particular developmental time point the

antidepressant drug imipramine starts to activate TrkB receptor in the mouse PFC and

hippocampus when injected intraperitoneally (30 mg/kg), whereas at the earlier

timepoints imipramine did not increase TrkB phosphorylation from the baseline. Also

imipramine-induced phosphorylation of CREB, which is activated downstream of the

TrkB-PLCγ pathway, appeared at P12. To demonstrate the role of BDNF in the TrkB

activation in vivo, we measured the baseline TrkB phosphorylation levels in BDNF

heterozygous knockout mice and BDNF knockout mice and found that at P11 the TrkB

phosphorylation was significantly lower in BDNF heterozygous mice and knockout mice

when compared to wild type mice. Intriguingly, in the adulthood there was no difference

in TrkB phosphorylation levels in BDNF conditional knockout or heterozygous knockout

mice and wild type mice, supporting the finding that BDNF is critical for the baseline

phosphorylation of TrkB at early postnatal age but dispensable in the adulthood.

To examine what could underlie the developmental switch in the TrkB responsiveness to

BDNF and ADs we conducted several experiments. The expression of the truncated

isoform of TrkB increases at the same developmental timepoint when the responsiveness

of TrkB to BDNF diminishes (Fryer et al., 1996). We used transgenic mice lacking the

TrkB.T1 receptor to examine the role of TrkB.T1 receptor in the TrkB responsiveness to

BDNF and ADs (III). The phosphorylation of TrkB in response to BDNF decreased

similarly in the TrkB.T1 KO mice microslices as in wild type mice microslices, indicating

that TrkB.T1 receptor expression does not regulate the activation of the full-length TrkB

receptor by exogenous BDNF. Also in adult TrkB.T1 KO mice imipramine induced TrkB

phosphorylation normally.

Chronic treatment with fluoxetine has been shown to promote neuronal plasticity and

restore more immature developmental stage in the adult rodent brain (Karpova et al.,

2011; Maya Vetencourt et al., 2008). We tested if chronic fluoxetine treatment (21 days)

46

could restore the TrkB responsiveness to BDNF in adult mice but no difference between

fluoxetine-treated and saline-treated animals was observed, implicating that the

juvenating effects of fluoxetine were not enough to restore the responsiveness of TrkB to

ex vivo BDNF (III).

By using a cell-free kinase assay we investigated if TrkB receptor itself had undergone

some structural modifications during development that could explain the decrease in

BDNF responsiveness (III). BDNF stimulation of cell-free lysates from adult brain in the

presence of ATP induced TrkB phosphorylation, demonstrating that the receptor can still

be activated by BDNF. In addition, NGF ex vivo stimulation could readily induce TrkA

activation at P24 suggesting that the ability of neurotrophins to penetrate the tissue is

not the reason for the reduced responsiveness of TrkB to BDNF and that the

developmental regulation may be a specific trait for BDNF.

5.4 Isoflurane activates TrkB signaling, enhances synaptic plasticity and

induces antidepressant-like behavior (IV)

Volatile anesthetic isoflurane has previously been shown to relieve depression symptoms

in treatment-resistant depressed patients (Langer et al., 1985, 1995; Weeks et al., 2013).

We examined the ability of isoflurane to affect BDNF-TrkB signaling and found that

isoflurane anesthesia readily activates TrkB receptor by inducing phosphorylation of the

catalytic domain Y706/7 and PLCγ binding site Y816 (but not Shc binding site Y515) of

TrkB (IV). In the prefrontal cortex and hippocampus of anesthetized mice

phosphorylations of CREB, Akt (not hippocampus), mTOR (not hippocampus), P70S6K,

4EBP-1 and GSK3beta were significantly increased. These TrkB downstream signaling

molecules have been previously shown to be phosphorylated by conventional

antidepressant drugs and especially by rapid-acting antidepressant ketamine (Li et al.,

2010; Rantamäki et al., 2007; Saarelainen et al., 2003). Interestingly, similarly to

antidepressant drugs, isoflurane does not induce TrkB phosphorylation in the

hippocampus of mouse pups at P8 (Fig. 5).

Fig 4. Isoflurane anesthesia did not induce TrkB activation in the prefrontal cortex or hippocampus of P8-9 wild type mice pups. Abbreviations: CTRL, control treatment; ISO, isoflurane treatment 15 min; PFC, prefrontal cortex; HC, hippocampus; TrkB.FL, full-length TrkB receptor.

47

The activation of TrkB by isoflurane occurs quickly, within few minutes (IV). When

measured 15 minutes after the end of the anesthesia the phosphorylation of TrkB has

returned close to the baseline indicating that the phosphorylation is transient. Activation

of the signaling molecules seems to be regulated in a similar manner, since the activation

of the mTOR and CREB occurs already in 2 minutes. Pretreatment with AMPA receptor

antagonist NBQX did not affect the ability of isoflurane to activate TrkB receptor and the

downstream signaling molecules, suggesting that the effects of isoflurane on TrkB

signaling are not mediated via AMPA receptors.

Importantly, isoflurane activated TrkB receptors also in the hippocampus of BDNF

conditional knockout mice, suggesting that TrkB activation by isoflurane occurs via

transactivation and is not mediated by BDNF (IV). To investigate the putative

mechanisms underlying the isoflurane-induced TrkB transactivation we conducted a

mass spectrometry analysis of proteins interacting with TrkB in the adult brain. We

found that PSD93, a synaptic scaffolding protein, interacts with TrkB. Since volatile

anesthetics have been shown to promote disruption of PSD93 and PSD95 interaction

from NR2B-subunits of NMDA receptors in vitro (Fang et al., 2003), we investigated if

isoflurane regulates the interaction between PSD93 and TrkB. We stimulated RN33

(immortalized raphe nuclei neuronal precursor cell line) cells with isoflurane and found

that isoflurane dose-dependently promoted disruption of TrkB-PSD93 interaction.

Moreover, reducing PSD93 expression in RN33 cells with siRNA increased the basal

TrkB phosphorylation levels, suggesting that releasing TrkB from the PSD93 protein

complex facilitates its activation. Interestingly, naïve PSD93 knockout animals showed

antidepressant-like behavioral effect in the FST.

To further elucidate the mechanisms that could be involved in the TrkB activation by

isoflurane we examined the Src family kinases. Previously, the Src family kinases have

been shown to be involved in the TrkB transactivation (Rajagopal and Chao, 2006). In

vitro, isoflurane treatment increased association of the activated Fyn with TrkB and

pretreatment with PP1 (protein phosphatase 1; Src inhibitor) abolished the effects of

isoflurane on TrkB phosphorylation (IV).

Since the signaling pathways activated by isoflurane have been linked with

synaptogenesis, cell survival and plasticity we examined the effects of isoflurane

anesthesia on LTP in Schaffer collaterals in hippocampus. Indeed, we found that in slices

from mice that were anesthetized for 30 minutes 24 hours before the electrophysiological

recordings the LTP was enhanced (IV). Interestingly, the LTP enhancement could be

blocked with picrotoxin pretreatment, suggesting that GABAergic component is

involved. Moreover, we found that GABAergic excitability is increased at this time point

and this finding was supported by increased FosB immunoreactivity (marker of neuronal

activity) in GABAergic cells of CA1 area of the hippocampus.

The increase in LTP was not, however, accompanied by an increase in hippocampal

dendritic spine number (IV). In addition, even though signaling pathways linked with

synaptogenesis are clearly activated in the PFC during the anesthesia, no significant

differences were seen in dendritic spine number or morphology in the PFC either.

TrkB signaling has been implicated in the antidepressant-like behavioral effects in the

FST. Since isoflurane robustly activates TrkB signaling, we tested if isoflurane produces

a behavioral response in the FST (IV). Indeed we found that isoflurane decreased the

48

immobility time indicating an antidepressant-like phenotype. Interestingly, this

behavioral response was abolished in the TrkB.T1 overexpressing mice suggesting that

TrkB signaling is necessary for the behavioral effects of isoflurane in the rodents.

To further study the behavioral effects of isoflurane we used the learned helplessness

model. Isoflurane produced a decrease in the escape latency if it was administered 24h

after the pretest and the actual test was conducted 6 days after the anesthesia indicating

an antidepressant-like effect (IV). We also examined the ability of isoflurane to

counteract the effects of chronic neuropathic pain on behavior of sciatic nerve cuffed

mice. After 8 weeks of sciatic nerve cuffing the mice showed increased latency to eat the

pellet in the novelty suppressed feeding test. Markedly, the anxiodepressive behavioral

phenotype was normalized with a single isoflurane anesthesia. These data implicate that

isoflurane produces antidepressant-like behavioral phenotype and may have rapid and

long-lasting antidepressant effects similar to ketamine.

49

6. DISCUSSION

TrkB neurotrophin receptor is an interesting target for drug development. The receptor

is able to activate downstream signaling cascades that facilitate neuronal excitability and

regulate translation and transcription of proteins promoting neuronal survival,

proliferation, differentiation and plasticity (Huang and Reichardt, 2003). Impaired

brain plasticity has been linked to many brain disorders, including depression, stroke

and neurodegenerative disorders, and promoting TrkB activity could be beneficial in

these conditions (Castrén and Rantamäki, 2010). Delivering exogenous BDNF to the

patients is difficult, since the neurotrophin cannot reach the brain if taken orally and

even injecting it directly to the brain does not guarantee its efficacy because of its poor

tissue penetrance (Mufson et al., 1994). Developing drugs that directly target the TrkB

receptor would be a possible approach to circumvent these issues.

We developed an in situ ELISA method that is suitable for screening of TrkB activators

and inhibitors (I). The in situ ELISA allows skipping of some laborious parts of the

conventional ELISA protocol and provides a possibility for high throughput screening,

also for industrial purposes. We showed that the phosphoTrk ELISA works in 384 well

plate format and applying it even to 1536 well plate format could be possible. Previously,

primary sensory neurons have been cultivated on ELISA plate (Balkowiec and Katz,

2000), and our purpose was to further develop the in situ ELISA to be suitable for

primary neurons, however, finding optimal conditions to support the survival of the

primary neurons in the ELISA plate for long enough was not yet possible.

The in situ ELISA method can also be used for discovering compounds that facilitate the

effects of BDNF. A compound that would enhance the effects of endogenous BDNF would

be physiologically relevant and allow more specific targeting of the drug effect. Activating

TrkB signaling randomly all over the brain could promote risk for epileptogenic activity

and randomly strengthen synapses creating more “noise” in the neuronal networks. Yet,

activation of TrkB receptor independently of BDNF could be useful especially in the

situations where the expression or release of BDNF is reduced, such as in patients with

BDNF met/met polymorphism.

Intriguingly, TrkB activating drugs already exist and are widely used by millions of

people – the antidepressant drugs. It has been previously demonstrated that

antidepressant drugs can activate TrkB receptor, at least in the rodent brain (Rantamäki

et al., 2007; Saarelainen et al., 2003). The clinical efficacy of different ADs is fairly

similar and their primary target in the brain are the widely projecting monoaminergic

systems. Even though the ADs primarily target the monoamines, they have effects that

are not directly mediated via the monoaminergic system. Especially the fact that ADs

require weeks before their antidepressive effects in humans appear, even though their

effects on the monoamines are acute, has evoked interest in examining the effects of

these drugs on targets outside the monoaminergic systems. The neurotrophin and

network theories of depression and antidepressant action suggest that brain plasticity is

involved in the pathophysiology of depression and in antidepressant action (Castrén and

Hen, 2013; Duman and Monteggia, 2006). One of the mediators of the plasticity-

inducing effects of ADs is BDNF, which acts through TrkB (Karpova et al., 2011; Maya

Vetencourt et al., 2008). We examined the mechanism of antidepressant-induced TrkB

50

activation and showed that it occurs independently of BDNF and monoamines,

suggesting that ADs transactivate TrkB receptor (II).

Neuronal activity is an important regulator of TrkB expression and localization on cell

surface (Du et al., 2000; Merlio et al., 1993) but transactivation of the receptor could

occur also in the absence of increased neuronal activity. In addition to receptors on the

cell surface also receptors residing inside the cell could be transactivated (Schecterson

and Bothwell, 2010). Activation of the intracellular pool of the TrkB receptors could

theoretically produce stronger response than the BDNF-dependent activation of limited

amount of cell surface receptors. Transactivation of the intracellular receptors could also

promote activation of immature Trk receptors. AD (and isoflurane) treatment constantly

induced phosphorylation of an unknown 105 kDa protein that we assume to be an

immaturely glycosylated form of TrkB. Glycosylation of the receptor can inhibit ligand-

independent activation of the receptor, and it has been shown that ERK pathway is not

activated via the immaturely glycosylated TrkA receptor (Watson et al., 1999a). Since

ADs transactivate TrkB receptor and do not induce signaling via ERK pathway, this

further supports the possibility that immaturely glycosylated form of the receptor is

activated by these drugs. Furthermore, in humans an N-terminal truncated form of TrkB

receptor is expressed and it is not targeted to the membrane but can be phosphorylated

(Luberg et al., 2010).

In addition, it is not known if TrkB transactivation promotes retrograde transport of the

activated receptors toward the soma. The retrograde transport has been implicated to be

important for the survival-promoting effects of neurotrophins in the PNS (Watson et al.,

2001). Nonetheless, increased CREB phosphorylation indicates that proteins in the soma

are also activated by ADs and isoflurane (II, IV). Transactivation of the TrkB receptor by

ADs does not activate the Shc binding site (Y515) of the receptor (II) that is readily

phosphorylated upon BDNF binding to the receptor (Segal et al., 1996). In addition, ADs

did not induce Akt or ERK activation that are in the canonical neurotrophin signaling

pathway mediated via the Shc binding site (II). The lack of Y515 phosphorylation could

be related to the conformational properties of the receptor when transactivated or to the

possibly different subcellular compartment where the transactivated receptors are

localized. Antidepressant drugs, thus, do not induce completely similar TrkB activation

as BDNF.

In addition to conventional ADs we found that isoflurane anesthesia can induce TrkB

receptor phosphorylation in the adult rodent brain (IV). The TrkB phosphorylation was

induced also by other inhalation anesthetics, sevoflurane and halothane, suggesting that

the effect may be common to all anesthetics. Similarly to ADs, TrkB and CREB are

activated during isoflurane anesthesia (Rantamäki et al., 2007). The TrkB activation by

isoflurane occurs already in couple of minutes, which is more quickly than by ADs (II,IV).

The more rapid effect of isoflurane is probably related to the different kinetics of volatile,

inhalation anesthetic when compared to intraperitoneally injected drugs. Interestingly,

in contrast to ADs (Li et al., 2010), isoflurane can activate Akt and mTOR-P70S6K-

4EBP1 –signaling in the PFC (IV). This signaling pathway has been previously shown to

be involved in the antidepressant-like behavioral effects and the rapid synaptogenesis

enhancing effects of ketamine (Li et al., 2010). Even though ADs and isoflurane are able

to activate the TrkB receptors, the differences in the activated signaling cascades could

explain some of the disparities in their effects. Isoflurane for example did not regulate

51

the activity of EEF2 that has been previously shown to be involved in the behavioral

effects of ketamine (Autry et al., 2011). Moreover, AMPA receptor blockade by NBQX did

not prevent the isoflurane-induced signaling effects, suggesting that AMPA receptor-

mediated effects are not critically involved in the regulation of TrkB signaling by

isoflurane (IV). However, AMPA receptor has been crucially linked to the

antidepressant-like effects of ketamine (Li et al., 2010; Maeng et al., 2008; Zanos et al.,

2016).

The ability of ketamine to induce rapid increase in the number of dendritic spines has

been suggested to underlie its rapid antidepressant effects (Li et al., 2010, 2011). Even

though the synaptogenesis-related signaling is robustly activated by isoflurane, we were

not able to demonstrate any increase in dendritic spine number in isoflurane treated

mice when examined 24 hours after the anesthesia (IV). Moreover, activity-dependent

secretion of BDNF has been linked to the synaptogenesis-promoting effects of ketamine,

since the ketamine-induced increase in dendritic spines was abolished in BDNF

heterozygous knockout mice (Liu et al., 2012). The ability of isoflurane to transactivate

TrkB independently of BDNF could be involved in the differerential effects of isoflurane

and ketamine on spines. Futhermore, it would have been important to include ketamine

as a positive control in the experiments, since the effects of ketamine on dendritic spines

in previous studies have been demonstrated mainly in rats (Li et al., 2010, 2011) and we

used mice. In addition, stress pretreatment could have been required, since ketamine has

been shown to counteract the stress-induced downregulation in the spine number (Li et

al., 2011). Previously, however, general anesthesia has been shown to promote

synaptogenesis in rats only at certain developmental stage (~P16) but not in the

adulthood, supporting our findings on naïve adult animals (Briner et al., 2010, 2011; Roo

et al., 2009). The negative effects of anesthetics on dendritic spine density have been

demonstrated in rodents at early postnatal age (<P10) (Briner et al., 2011) and since the

developmental shift also in TrkB activation by isoflurane occurs around this same time

point, it is tempting to speculate that TrkB signaling could be involved in the

synaptogenesis-promoting effects of isoflurane.

The developmental shift in the TrkB response to BDNF and ADs occurs around P12 (III).

Even though in the adult rodent brain ADs induce TrkB activation, they do not activate

TrkB receptors when injected to mice at early postnatal age (III). This same applies also

for isoflurane, since isoflurane anesthesia at P8-9 does not produce TrkB activation (Fig

5.). The developmental shift in the TrkB activation is not related to changes in the

structure of the receptor itself but could be explained by changes in the TrkB interacting

partners capable of regulating the activation and localization of the receptor (III). We

have not been able to demonstrate TrkB activation by ADs in cultured hippocampal or

cortical neurons and it is possible that the lack of proper interacting partners at the

neuronal cells derived from embryonic rat brain could be an explanation. Also the TrkB

transactivation by ADs may require functional neuronal network that would respond to

AD treatment in a complex manner recruiting e.g. G-protein coupled receptors, reducing

the activity of protein phosphatases or affecting general neuronal activity that would then

eventually lead to TrkB activation. Thus, TrkB transactivation by ADs may require

processes and mediators that emerge only after certain developmental stage.

The lack of TrkB activation in cell culture by drugs that can promote TrkB activation in

vivo complicates the study of the mechanisms of AD-induced TrkB activation in detail

52

using cultured cells. This also has to be taken into account when using the cell-culture

based in situ ELISA for screening of TrkB regulating drugs. Nonetheless, the maturation

of the cultured neurons (or other cells), the cell line used and general culture conditions

can affect the ability of different compounds to activate TrkB receptor. Previously e.g.

amitriptyline and 7,8-DHF have been shown to activate TrkB receptor in vitro (Jang et

al., 2009, 2010) and also in our studies we were able to detect isoflurane-induced TrkB

activation in RN33 cells (IV).

In addition to the developmental shift in the TrkB activation by ADs, the ability of

exogenous BDNF to activate TrkB receptor in ex vivo context is developmentally

regulated (III). Importantly, the reduction in BDNF-induced TrkB activation occurs at

the same time point when the AD effect appears (~P12). Changes in the interactome

could also explain the lack of BDNF effect if there are interacting proteins capable of

preventing the conformational changes of the receptor upon BDNF binding or the

autophosphorylation of the TrkB receptors. We did not examine in this study whether

BDNF injection directly to hippocampus of adult mice would increase TrkB activation.

The baseline TrkB activation in the brain of BDNF conditional knockout or heterozygous

knockout mice is comparable to wild type mice suggesting that BDNF is not regulating

the basal level of TrkB phosphorylation in the adulthood (III). In studies where BDNF is

injected to the brain, its ability to activate TrkB receptor is not normally investigated,

however, in the study by Guo et al. (2014) BDNF injection to adult rat hippocampus could

promote phosphorylation of the tyrosine 515 of TrkB. Thus, it is not known for sure if the

reduction in TrkB activation by BDNF is a special feature of the ex vivo treatment of brain

microslices and if it applies or not in vivo.

In addition to the activation of molecules linked to synaptogenesis, the enhanced

hippocampal LTP 24 hours after isoflurane anesthesia could be accompanied by

increased mushroom spine number. Yet, we did not find any difference in the spine

morphology between isoflurane and sham treated animals (IV). Interestingly, we found

that picrotoxin pretreatment could block the enhanced LTP by isoflurane, which

indicates that changes in the GABAergic system are involved in the effects of isoflurane.

This was further supported by the finding that the GABAergic excitability was enhanced

and the activity of the inhibitory interneurons was increased 24 hours after isoflurane

treatment. The explanation for the lack of changes in the excitatory spines could be that

the changes occur rather in the amount and/or strength of the inhibitory synapses than

in the excitatory synapses.

The changes in the neuronal network excitability could underlie the long-lasting

behavioral effects of isoflurane. The long-lasting effects of isoflurane on rodent behavior

were demonstrated in the learned helplessness model, where a single isoflurane

treatment produced an antidepressant-like effect when the test was conducted 6 days

after the treatment (IV). To show that isoflurane has more rapid effects than

conventional ADs we used novelty suppressed feeding test. The conventional ADs require

weeks of administration before they produce an effect in the novelty suppressed feeding

test (David et al., 2009), but a single isoflurane treatment only 12 hours before the test

was enough to normalize the behavior of the mice in the neuropathic pain model of

depression (IV). Probably already at this time point changes occur in the network

function of the isoflurane-treated animals that could underlie the behavioral phenotype.

53

Ketamine has also been suggested to activate TrkB receptor at subanesthetic dose and

the behavioral effect of ketamine in FST was abolished in TrkB conditional knockout

mice (Autry et al., 2011). We found that TrkB signaling is required also for the behavioral

effects of isoflurane in the forced swim test. If TrkB receptor activation is an important

mediator of the rapid antidepressant effects of ketamine and isoflurane, it is interesting

why conventional antidepressant drugs then require weeks before their effect arises even

though they induce TrkB phosphorylation already after single injection. Differences in

the downstream signaling and the possible rapid increase in BDNF translation have been

suggested to underlie the differential effects of conventional and rapid-acting ADs (Autry

et al., 2011; Li et al., 2010). In addition to antidepressant drugs and anesthetics, also

anticholinesterases galantamine and donepezil, used in the treatment of Alzheimer’s

disease, can induce TrkB, Akt and CREB activation in mouse hippocampus (Autio et al.,

2011). Thus, drugs with completely different mechanism of action and different

therapeutic effects produce TrkB activation. All these drugs do not produce

antidepressant effects in humans, suggesting that TrkB activation solely is not sufficient

to guarantee antidepressant efficacy.

We examined the behavioral effects of isoflurane in paradigms that are widely used to

elucidate the potential antidepressant-like effects of drugs. In general, it is impossible to

model depression thoroughly in rodents and the models are able to dissect only some

specific aspects of depression, e.g. anhedonia or coping in stressful situation. The ability

of drugs to alter the behavior of rodents in these tests allows examination of the critical

factors that underlie the drug-induced behavioral phenotype in question. These factors

can then be determined to participate in the effects that the drug mediates. However, it

is important to be cautious when claiming that the factors regulating rodent behavior are

crucial for the antidepressant effects in human patients. In addition, even though

isoflurane anesthesia produces similar changes in rodent behavior as antidepressant

drugs, to prove that isoflurane relieves depression in humans, and that the effect is rapid

and long-lasting, requires clinical studies.

One limitation of our study is that we examined the effects of isoflurane anesthesia only

on selected proteins downstream of TrkB receptor and on proteins previously linked with

the rapid-antidepressant effects of ketamine. Apparently, isoflurane can induce

phosphorylation of a wide range of proteins as was demonstrated by phosphoproteomic

analysis from isoflurane treated animals (Kohtala et al., 2016) suggesting that all the

effects are not specific to TrkB signaling pathways, but may be related to a more general

increase in protein phosphorylation. The induction of protein phosphorylation may be

dose-dependent, since subanesthetic dose of isoflurane did not induce similar increase

in protein phosphorylation (IV).

Most probably, the antidepressant effects of isoflurane are not induced only by BDNF or

TrkB but require co-expression of other molecules involved in the regulation of neuronal

function and plasticity. The effects of conventional ADs and rapid-acting antidepressants

on these molecules probably differ significantly. Ketamine and isoflurane for example

robustly affect the NMDA and GABA receptors that directly regulate the neuronal

network excitability whereas the effects of conventional antidepressant drugs, mediated

via e.g. elevated levels of serotonin, are modulatory and slower in action. The ability of

ketamine to rapidly affect synaptic function and plasticity has been suggested to underlie

its rapid antidepressant effects (Duman et al., 2016). In general, drugs that would

54

produce rapid effects on synaptic functions in neuronal circuits involved in mood

regulation could theoretically have antidepressant potential. For example psilocybin,

which induces psychedelic effects and acutely modulates the functional connectivity of

the brain, was recently shown to have antidepressant effects in human patients (Carhart-

Harris et al., 2013, 2016). One of the problems related to the therapeutic use of ketamine

is that the antidepressant effect in human patients is often transient (Krystal et al., 2013).

Even though acute and rapid regulation of the mood is possible by ketamine, stabilizing

the functions of the mood networks into a beneficial state appears to be difficult.

The ability of single isoflurane anesthesia to produce robust effects on the signaling

pathways and to induce changes in the neuronal excitability, synaptic function and

rodent behavior may cause problems in experimental procedures where isoflurane

anesthesia is used. This is important to take into account when planning experiments

where anesthesia is required.

The effects of antidepressants and isoflurane on TrkB activation are especially

interesting in the context of neuronal plasticity. Chronic treatment with antidepressant

drugs can promote brain plasticity via BDNF–dependent mechanism (Karpova et al.,

2011; Maya Vetencourt et al., 2008). ADs induce TrkB activation already after single

injection, however, increase in BDNF protein requires chronic treatment (Nibuya et al.,

1995; Rantamäki et al., 2007; Saarelainen et al., 2003). It is possible that the TrkB

transactivation is not enough to promote the critical period like plasticity demonstrated

with fluoxetine, since in the study of Karpova et al. (2011) the effects of ADs and

extinction training on fear removal were abolished in BDNF heterozygous knockout

mice, even though in these mice ADs induce TrkB receptor activation similarly to wild

type mice (II). The possible explanation could be that TrkB receptors are activated

differentially by ADs when compared to BDNF, which could result in lack of activation

of some important downstream mediators required for the induction of plasticity. BDNF

can also have off-target effects that are not yet characterized. Currently, the role of TrkB

receptor in the mechanism of action of antidepressant drugs in humans is not known,

and solid evidence about the ability of antidepressant drugs to promote brain plasticity

in humans is lacking.

55

7. CONCLUSIONS

In this thesis we aimed to examine the mechanisms of antidepressant-induced TrkB

activation and elucidate the neurobiological basis for the antidepressant effects of

isoflurane anesthesia. In our experiments we were able to demonstrate that TrkB

receptor activation by antidepressant drugs can occur independently of BDNF in vivo.

The exact mechanism of how antidepressant drugs promote TrkB activation, however,

requires further characterization. The transactivation of TrkB receptor by antidepressant

drugs seems to differ from BDNF-induced TrkB activation since there are differences in

the phosphorylation of the tyrosines of TrkB and activation of the downstream signaling

molecules (Fig 5). Moreover, the TrkB receptor transactivation by ADs appears only after

certain developmental stage (P12 in mice), when TrkB responsiveness to BDNF is

reduced, indicating that the effects of ADs during development may differ from those in

the adult. The ability of isoflurane to promote brain plasticity and to activate signaling

pathways linked to the mechanisms of action of conventional antidepressant drugs and

ketamine supports the possibility that isoflurane anesthesia could be a potential

treatment option for depressed patients.

FIG 5. Activation of TrkB receptors by BDNF, antidepressant drugs and isoflurane. BDNF induces phosphorylations of Y515, Y706/7 and Y816 of TrkB, whereas antidepressant drugs and isoflurane do not phosphorylate Y515. BDNF activates TrkB downstream signaling pathways including Akt, ERK and PLCγ1, whereas antidepressant drugs activate only the PLCγ1 signaling. Isoflurane induces signaling via PLCγ1 and Akt. Antidepressant drugs and isoflurane activate the TrkB receptor via transactivation. The TrkB responsiveness to BDNF (ex vivo) and antidepressant drugs/isoflurane (in vivo) is developmentally regulated so that the responsiveness to BDNF decreases around postnatal day 12 and at this same timepoint the TrkB responsiveness to antidepressant drugs/isoflurane appears. Abbreviations: BDNF, brain-derived neurotrophic factor; AKT, protein kinase B; ERK, extracellular signal-regulated kinase; PLCγ1, phospholipase C gamma 1; pTrkB, phosphorylated TrkB receptor; P0, postnatal day 0; P12, postnatal day 12; Y, tyrosine.

The main conclusions are:

1. Antidepressant drugs transactivate TrkB receptor.

2. TrkB receptor activation by antidepressant drugs is developmentally regulated.

3. Isoflurane anesthesia induces molecular, functional and behavioral effects

similar to those of conventional ADs and ketamine.

56

ACKNOWLEDGEMENTS

The work of this thesis was carried out in the Neuroscience Center during years 2010-

2016 and has been financially supported by Doctoral Programme Brain & Mind, Orion-

Farmos Research Foundation, Sigrid Jusélius foundation, Academy of Finland and

European Research Council.

My deepest gratitude goes to my supervisors docent Tomi Rantamäki and professor Eero

Castrén. I always remember the first time I contacted Tomi by email to ask about a

possibility to do my Master’s thesis in the Trophin Lab. I thought he will probably never

answer to my email, but he did - in half an hour. In couple of hours we had already agreed

that I will come to meet him and possibly start my Master’s thesis project in the lab.

When we met, he talked about TrkB, tPA, proBDNF, BDNF, MMPs, western blot, ELISA,

zymography, and I understood nothing. During these years, however, I have learnt

something (well, a lot) and that is mainly because of Tomi. I am grateful to him that he

has supported me all the time during these years and believed in me and, importantly,

made me believe in myself. I wish I have absorbed at least a bit of Tomi’s enthusiasm,

intelligence, and diligence during these years. From a graduate student’s point of view I

have been very lucky since as a supervisor Tomi has always been available for discussion

- and for beers! I am as grateful to my other supervisor, Eero. Eero is a visionary and his

vast knowledge about neuroscience (and many other things) has always impressed me.

It has been such a privilege to be able to work in his lab and learn from him. In the darkest

moments of the lab work, Eero has an incredible ability to make a person feel again

enthusiastic and ambitious about science. It is impossible to leave his office without

feeling that the things that I’m doing actually make sense.

I want to thank the reviewers of my thesis, associate professor Annakaisa Haapasalo and

docent Mikko Airavaara, who critically evaluated the thesis during the summer. They

made me think many things from a different point of view and their comments were of

great importance in significantly improving the thesis manuscript. In addition, I want to

sincerely thank Giuseppe Cortese, who revised the English language of the thesis.

I am grateful to Professor Moses Chao for accepting the invitation to act as my opponent

in the public defense of this thesis.

Definitely, I have not done alone the work presented in this thesis, so all the co-authors

of the publications and the isoflurane manuscript are thanked for the great work that

they have done. Without you I could not have finished my PhD.

The people in the Trophin lab have changed completely during my thesis project, only

Outi, our irreplaceable lab technician has been here longer than me. I want to thank Outi

for always being interested in how I am doing. I think that a modified sentence from

Tomi’s thesis acknowledgements fits here well: “Your effort in the lab can only be

underestimated”. I cannot even imagine the amount of suffering that I would have

needed to survive if you had not been there ordering reagents, helping me to find things

in the lab and teaching me how to use ultracentrifuge or pH meter or electronic pipette

or… Also our other technician, Sulo, is thanked for always offering his help to me and

especially for his dark humor that has cheered me up - although I’m sure that was not his

intention! Our previous lab technician Hanna Jr. is also thanked for all the help and all

the laughs.

57

All the past and present members of the Trophin lab are warmly thanked for all the help,

discussions, ass-kicking, peer-support and great moments in and outside the lab. Ettore,

Yumiko, Marie, Henri, Liisa, Jesse and Ramon – I still miss you all. The two marvelous

people sitting next to me in the office, Anna and Juzoh, are thanked for sharing with me

in addition to the office also science, laughs, desperate moments, happiness, chocolate,

cookies, and lunch. I’m thankful of course also to the other current members of our lab

Frederike, Merve, Plinio, Carol, Madhu and all the students, for making this such a

pleasant place to work, we have a great team! In addition, I want to thank all the other

members of the Neuroscience Center and the laboratory animal facilities that I have

worked and/or partied with!

I am grateful to the members of HKV:n aikuisten pikajuoksuryhmä for the Tuesday

evening sprints that allow me to think about something else than TrkB - namely Trk &

field – for a while! The members of my extended family in Crossfit Herttoniemi are

thanked for sharing with me the sweatiest, and some of the funniest, moments of my life.

Especially I want to thank Nyytit (Maija, Miia, Mikko, Perttu, Tatu and Pauli) for all the

bönthö and “support” during the thesis writing. My dearest and oldest friends, Paula,

Annemari and Heli, are thanked for always being there for me.

Jussi is thanked for everything. Without your help, support and encouragement this

thesis would still be in preparation. I have learnt so much from you about myself, life and

science that I would be completely different person if I had not known you.

I want to warmly thank my family, isä, äiti, JP and Ville, for all the love and support.

August 2016, couple of days after back squatting 120 kg, just before the thesis should be sent to print,

Hanna

58

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